Methods and compositions for reducing cardiac damage and other conditions

ABSTRACT

Endoglin has been identified to play a functional role as a regulator of TGFβ1 signaling, particular in TGFβ1-mediated calcineurin expression. The present invention features methods of reducing cardiac damage, particularly in a subject undergoing chemotherapy or radiation therapy by administering a composition that inhibits endoglin activity. The present invention also features methods of treating autoimmune diseases, inflammatory diseases, organ transplantation, and conditions association with oxidative stress related to TGFβ1-mediated calcineurin expression and reactive oxygen species (ROS) production by administering a composition that inhibits endoglin activity. The present invention also features methods of treating fibrotic diseases by administering a composition that inhibits endoglin activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 15/022,663, now U.S. Pat. No. 10,214,590,filed Mar. 17, 2016, which is a National State Entry of InternationalPatent Application No. PCT/US2014/056313, filed Sep. 18, 2014, whichclaims priority to U.S. Patent Application No. 61/880,551, filed Sep.20, 2013, the contents of which are hereby incorporated by reference intheir entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under grant HL094909awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 13, 2019, isnamed 00398-536003_Sequence_Listing_5.13.19_ST25 and is 10,829 bytes insize.

BACKGROUND OF THE INVENTION

This invention relates to methods of reducing cardiac damage,particularly cardiac damage as a result of chemotherapy or radiationtherapy. The invention also relates to the treatment of autoimmunediseases, fibrosis, inflammatory diseases, organ transplantation, andconditions associated with oxidative stress.

Right ventricular (RV) failure is a major determinant of morbidity andmortality for millions of individuals worldwide who suffer from lungdisease or heart failure (McLaughlin et al., J Am Coll Cardiol.53:1573-1619, 2009, Haddad et al., Circ Heart Fail. 4:692-699, 2011). RVfailure is commonly a direct consequence of RV pressure overload (RVPO).Recent data confirms that elevated pulmonary artery systolic pressuresare inversely associated with RV ejection fraction and directly relatedto increased mortality in both lung disease and left heart failure(Benza et al., Circulation. 122:164-172, 2010, Bursi et al. J Am CollCardiol. 59:222-231, 2012).

TGFβ1 is a powerful cytokine that governs cardiac fibrosis and signalsthrough a heteromeric receptor complex comprised of a Type IIligand-binding receptor, a Type I activin-like kinase signalingreceptors, and Type III accessory receptors, including endoglin. Uponactivation, this receptor complex phosphorylates downstream effectorproteins known as Smads (canonical pathway) or mitogen activated proteinkinases (noncanonical pathway), including extracellular regulated kinase(ERK) (Leask, Cardiovasc Res. 74:207-212, 2007, Massague, Annu RevBiochem. 67:753-791, 1998). Specifically, TGFβ1-induced phosphorylationof Smads-2/3 and ERK promotes Type I collagen synthesis and fibroblastproliferation (Kuwahara et al., Circulation. 106:130-135, 2002).

The calcium-dependent serine/threonine phosphatase, calcineurin, isanother critical mediator of maladaptive cardiac remodeling, defined byexcessive fibrosis and hypertrophy. Studies have shown that calcineurinincreases expression of the canonical transient receptor protein channel6 (TRPC-6), which triggers calcium influx and subsequent calcineurinactivation, thereby setting up a self-propagating mechanism forpathologic hypertrophy, fibrosis, and increased mortality in heartfailure. Noncanonical TGFβ1 signaling through TRPC-6 was reported to bean important stimulus for calcineurin-mediated alpha-smooth muscle cellactive (α-SMA) expression, a marker of myofibroblast transformation anda critical component of cardiac fibrosis.

While it was recently reported that reduced endoglin expression limitsleft ventricular (LV) fibrosis and improves survival in a murine modelof LV failure (Kapur et al., Circulation. 125:2728-2738, 2012), less isknown about the functional role for endoglin in the RV and generally inorgan fibrosis. Accordingly, there is a need to develop new targets forpromoting RV cardiac remodeling for the treatment of heart failure.There is also a need to develop new targets for reducing organ fibrosis,such as, lung disease, and kidney disease, as well as new therapeuticapproaches to prevent organ, heart, and other fibrosis related morbidityand mortality.

SUMMARY OF THE INVENTION

As described in detail below, endoglin was shown to be a centralcomponent of fibrogenic signaling in the RV and a positive regulator ofTGFβ1-induced calcineurin/TRP expression. Given the importance ofcalcineurin in adaptive and maladaptive cardiac remodeling, targetingendoglin will result in reduced cardiac damage and improved survival.Furthermore, as endoglin was shown to modulate fibrotic signalingthrough the TGFβ1 pathway, a major signaling pathway in the initiationand progression of fibrogenesis, targeting endoglin provides atherapeutic approach for treatment of fibrotic diseases and preventionof fibrosis related morbidity and mortality. The inventors havediscovered that reducing expression or activity of the membrane-boundreceptor form of endoglin limits TGFβ1 signaling, not only in the heart,but in other organs (e.g., lung and kidney), thus resulting in a methodfor reducing organ fibrosis and improving survival.

Accordingly, in a first aspect, the invention features a method ofreducing cardiac damage in a subject undergoing chemotherapy orradiation therapy, the method including administering to the subject atherapeutically effective amount of a composition that inhibits endoglinactivity, wherein administration of the composition is begun prior to orconcurrently with the start of chemotherapy or radiation therapy orfollowing the development of chemotherapy- or radiation therapy-inducedheart disease or heart failure. The composition may include an antibody,an antigen-binding fragment thereof, an RNAi agent, or a solublepolypeptide. In one embodiment, the antibody or antigen-binding fragmentspecifically inhibits endoglin activity or the antibody orantigen-binding fragment is an antagonist of the endoglin receptor. In asecond embodiment, the polypeptide includes the amino acid sequence ofsoluble endoglin or an endoglin signaling-inhibitory fragment or analogthereof. In a third embodiment, the polypeptide is a protease, where theprotease is matrix metalloproteinase 14 (MMP-14), an active fragmentthereof, or includes an amino acid sequence having at least 80% identityto the amino acid sequence of MMP-14 having protease activity.

In particular embodiments, administration of the composition reduces,repairs, or remodels cardiac damage. In other embodiments,administration of the composition results in a reduction, repairing, orremodeling of cardiac fibrosis, ventricular hypertrophy, or improvementin blood vessel growth. In particular aspects, the reduction, repairing,or remodeling of cardiac damage in the subject is measured by animprovement in a cardiovascular parameter compared to a subjectundergoing chemotherapy alone, where the cardiovascular parameter isselected form the group consisting of: end-diastolic volume,end-systolic volume, stroke volume, ejection fraction, heart rate, andcardiac output. In yet another embodiment, administration of thecomposition results in reduced levels of reactive oxygen species (ROS),reduction of TRP expression and/or activity, reduction of α-SMAexpression and/or activity, or reduction of calcineurin expressionand/or activity. Preferably, administration of the composition resultsin reduction of expression of one or more members of the TRP family,such as, TRPC, TRPM, or TRPV expression (e.g., TRPC-6, TRPM3, or TRPV2expression). In another embodiment, the chemotherapy includesadministration of a chemotherapeutic agent selected from the groupconsisting of: an alkylating agent, an anthracycline, an epothilone, ahistone deacetylase inhibitor, an inhibitor of topoisomerase I, aninhibitor of topoisomerase II, a cytoskeletal disruptor, a kinaseinhibitor, a monoclonal antibody, a peptide antibiotic, a nucleotideanalog/precursor analog, a platinum-based agent, a retinoid, and a vincaalkaloid.

In another aspect, the invention features a method of treating ortreating prophylactically a subject having an autoimmune disease, havinga non-autoimmune inflammatory disease, or having undergone organtransplantation, the method including administering to the subject atherapeutically effective amount of a composition that inhibits endoglinactivity. In certain embodiments, the composition is administered inaddition to an immunosuppressive agent. In other embodiments, thecomposition is administered prior to administration of theimmunosuppressive agent. In yet another aspect, the invention features amethod of treating a condition associated with oxidative stress in asubject in need thereof, the method including administering to thesubject a therapeutically effective amount of a composition thatinhibits endoglin activity. In certain embodiments, the composition isadministered in combination with a second agent, where the second agentis an anticancer/antiproliferative drug, a cardiovascular drug, or ananti-neurodegenerative drug. In other embodiments, the conditionassociated with oxidative stress is selected from the group consistingof: reperfusion injury, wound healing, toxic hepatitis, viral hepatitis,cirrhosis, chronic hepatitis, idiopathic pulmonary fibrosis, chroniclung disease, oxidative stress from dialysis, renal toxicity, kidneyfailure, ulcerative colitis, bacterial infection, viral infections,upper respiratory tract diseases, organ fibrosis, skin fibrosis,scleroderma, oxidative stress due to sun damage, and cancer. Inparticular embodiments, the condition associated with oxidative stressis a chronic condition. In some embodiments, the chronic condition ischronic organ disease, selected from the group consisting of: chroniclung disease, chronic obstructive pulmonary disease, chronic viralhepatitis, chronic renal disease, chronic pancreatitis, chronicprostatitis, chronic inherited bleeding disorders, and chronic bonedisease. In certain aspects, the administration of the compositionreduces the levels of reactive oxygen species (ROS).

In a final aspect, the invention features a method of treating afibrotic disease in a subject in need thereof, the method includingadministering to the subject a therapeutically effective amount of acomposition that inhibits endoglin activity. In some embodiments, thefibrotic disease is selected from the group consisting of idiopathicpulmonary fibrosis, organ fibrosis, interstitial lung disease, skinfibrosis, diabetic nephropathy, liver fibrosis, liver cirrhosis,nonalcoholic steatohepatitis (NASH), rheumatoid arthritis,fibrosarcomas, keloids and hypertrophic scars, arteriosclerosis, kidneydisease, macular degeneration, retinal and vitreal retinopathy, surgicalcomplications, chemotherapeutic drug-induced fibrosis, radiation-inducedfibrosis, accidental injury, burns, local scleroderma, and systemicscleroderma. Preferably, the fibrotic disease is idiopathic pulmonaryfibrosis. In some embodiments, the composition is administered with anantifibrotic agent, selected from the group consisting of:pentoxyphiline, tocopherol, vitamin E, pioglitazone, INT 747,peginterferon 2b, infliximab, ribavirin, glycyrrhizin, candesartan,losartan, irbesartan, ambrisentan, FG-3019, warfarin, insulin,colchicines, peginterferon 2a, etanercept, pirfenidone, nintedanib, andIL-10. In particular embodiments, administration of the compositionreduces the levels of ROS, collagen expression, or promotes tissueremodeling.

In all embodiments of the invention, the composition that inhibitsendoglin signaling is formulated for oral, parenteral, cutaneous,subcutaneous, topical, transdermal, ocular administration, or byinjection, inhalation, or direct contact with the nasal or oral mucosa.In other embodiments of all of the above inventions, the compositioninhibits TGFβ1-mediated endoglin activity or calcineurin-mediatedendoglin activity. In yet another embodiment of the above inventions,the administration of the composition further provides cardiacprotection in the subject.

Definitions

By “administration prior to” is meant administration of a composition ofthe invention in a therapeutically effective amount before the start ofchemotherapy or radiation therapy (e.g., 4 weeks prior, 3 weeks prior, 2weeks prior, 1 week prior, 6 days prior, 5 days prior, 4 days prior, 3days prior, 2 days prior, 1 day prior, less than 24 hours prior (e.g.,less than 23, 20, 19, 18, 17, 16, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 hours,or 1 hour) to the start of chemotherapy or radiation therapy.

By “administration concurrently with” is meant administration of acomposition of the invention in a therapeutically effective amount withthe start of chemotherapy or radiation therapy (e.g., less than 2, 6,12, 18, or 24 hours after the start of chemotherapy or radiationtherapy. Alternatively, “administration concurrently with” can meanbetween the first and second doses of chemotherapy or radiation therapy.

By “chemotherapy” is meant treatment of a disease by administering anagent (e.g., a small molecule, an antibody, or an antigen-bindingfragment thereof) that reduces or reverses the growth of cancer cells(e.g., destroys cancerous tissue).

By “chronic” is meant the state of human health condition or diseasethat is persistent or otherwise long-lasting in its effects (e.g.,course of condition or disease that last for more than three months).Chronic conditions or diseases often lead to morbidity and/or mortality.Examples of chronic conditions and diseases include but are not limitedto cancer, blindness, Alzheimer's disease, Parkinson's disease,deafness, mental illness, chronic pain syndromes, and those describedherein, for example, chronic lung disease, chronic obstructive pulmonarydisease, chronic viral hepatitis, chronic renal disease, chronicpancreatitis, chronic prostatitis, chronic inherited bleeding disorders,or chronic bone disease.

By “subject” is meant a human or non-human animal (e.g., a mammal).

By “soluble endoglin” is meant a polypeptide that includes theextracellular domain of endoglin, but does not include the transmembraneor cytoplasmic domains of endoglin and has the ability to decreaseTGFβ1-mediated activation of the endoglin receptor.

By “soluble endoglin fragment” is meant a fragment of at least 4, 5, 6,8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 100, 125, 150, 175, 200, 225,250, 300, 350, 400, or 450 amino acids of soluble endoglin.

By “at least 80% identity” is meant a polypeptide or polynucleotidesequence that has the same polypeptide or polynucleotide sequence,respectively, as a reference sequence, or has a specified percentage ofamino acid residues or nucleotides, respectively, that are the same atthe corresponding location within a reference sequence when the twosequences are optimally aligned. For example, an amino acid sequencethat is “at least 80% identical” to a reference sequence has at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to thereference amino acid sequence. For polypeptides, the length ofcomparison sequences will generally be at least 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250,300, or 350 contiguous amino acids (e.g., a full-length sequence). Fornucleic acids, the length of comparison sequences will generally be atleast 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25 contiguous nucleotides (e.g., the full-length nucleotide sequence).Sequence identity may be measured using sequence analysis software onthe default setting (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705). Such software may matchsimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications.

By “treating” a disease, disorder, or condition in a subject is meantreducing at least one symptom of the disease, disorder, or condition byadministrating a therapeutic agent to the subject.

By “treating prophylactically” a disease, disorder, or condition in asubject is meant reducing the frequency of occurrence of or reducing theseverity of a disease, disorder or condition by administering atherapeutic agent to the subject prior to the onset of disease symptoms.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J show that reduced endoglin expression improves survival andlimits calcineurin activity after right ventricular pressure overload.FIGS. 1A-1B show levels of endoglin mRNA and protein expression in WTand Eng+/− mice after PAC (n=6/group). FIG. 1C shows Kaplan-Meiersurvival curves in WT and Eng+/− mice after PAC (n=12/group). FIG. 1Dshows right ventricular systolic pressure in WT and Eng+/− mice afterPAC (n=6/group). FIG. 1E shows right ventricular stroke volume in WT andEng+/− mice after PAC (n=6/group). FIG. 1F shows total body weight in WTand Eng+/− mice after PAC (n=6/group). *, p<0.05 vs Sham; †, p<0.05 vsWT vs. Eng+/− sham, ‡, p<0.05 Wt vs. Eng+/−PAC. FIG. 1G show histologicstaining (hematoxylin and eosin) of right ventricular (RV)cardiomyocytes in WT and Eng+/− mice after pulmonary artery constriction(PAC). FIG. 1H shows quantification of RV cardiomyocyte cross-sectionalarea. FIGS. 1I-1J show quantification of RV pSmad-3 and p-ERK1/2 proteinlevels in WT and Eng+/− after PAC. Representative western blots areshown below graphs (*, p<0.05 vs Sham; †, p<0.05 vs WT-PAC).

FIGS. 2A-2M show that reduced endoglin expression improves survival andlimits calcineurin activity after right ventricular pressure overload.FIGS. 2A-2B show representative histologic staining for RV collagenabundance in WT and Eng+/− mice after PAC. Quantification of RV fibrosisafter PAC is shown (n=6/group). FIG. 2C shows quantification of RV TypeI collagen protein levels in WT and Eng+/− mice after PAC (n=6/group). Arepresentative western blot is shown. FIG. 2D shows levels of activeTGFβ1 in RV protein lysates from WT and Eng+/− mice (n=6/group). FIGS.2E-2F show levels of RV calcineurin mRNA and protein in WT and Eng+/−mice after PAC (n=6/group). A representative western blot is shown.FIGS. 2G-2I show levels of RV MYH7, TRPC-6, and α-SMA mRNA expression inWT and Eng+/− mice after PAC (n=6/group). *, p<0.05 vs Sham; †, p<0.05vs. WT-PAC. FIG. 2J shows histologic staining (hematoxylin and eosin) ofright ventricular (RV) cardiomyocytes in WT mice treated with a N-Eng Abor IgG Ab after pulmonary artery constriction (PAC). FIG. 2K showsquantification of RV cardiomyocyte cross-sectional area. FIGS. 2L-2Mshow quantification of RV pSmad-3 and p-ERK1/2 protein levels in WT micetreated with a N-Eng Ab or IgG Ab after PAC. Representative westernblots are shown below graphs (*, p<0.05 vs Sham; †, p<0.05 vs. WT-PAC).

FIGS. 3A-3I show that neutralizing endoglin activity improves survivaland limits the development of cardiac fibrosis after right ventricularpressure overload. FIG. 3A shows Kaplan-Meier survival curves in WT micetreated with an IgG control antibody or N-Eng Ab after PAC (n=18/group).FIGS. 3B-3C show representative histologic staining for RV collagenabundance in IgG versus N-Eng Ab treated mice after PAC. Quantificationof RV fibrosis after PAC is shown (n=6/group). FIG. 3D showsquantification of RV Type I collagen protein levels in IgG versus N-EngAb treated mice after PAC (n=6/group). A representative western blot isshown. FIG. 3E shows quantification of RV calcineurin protein levels inIgG versus N-Eng Ab treated mice after PAC (n=6/group). A representativewestern blot is shown. FIGS. 3F-3H show levels of RV MYH7, TRPC-6, andα-SMA mRNA expression in IgG versus N-Eng Ab treated mice after PAC(n=6/group). *, p<0.05 vs. Sham; †, p<0.05 vs. WT+N-Eng Ab PAC. FIG. 3Iis Western blots showing protein levels of pSmad-3 and pERK-1/2 in RVFBand LVFB stimulated with TGFb1 in the presence and absence of increasingconcentrations of N-Eng Ab.

FIGS. 4A-4E show that reduced endoglin activity limits calcineurinexpression and myofibroblast conversion in right ventricularfibroblasts. FIGS. 4A-4B show calcineurin and α-SMA mRNA levels infibroblasts from the right (RVFB) and left (LVFB) ventricles of WT andEng+/− mice before and after TGFβ1 stimulation. FIG. 4C is a set ofrepresentative western blots showing calcineurin-SMA levels after TGFβ1stimulation in RVFB and LVFB from WT and Eng+/− mice. FIGS. 4D-4E showquantification of calcineurin and α-SMA protein levels in RVFB and LVFBstimulated with TGFβ1 in the presence and absence of increasingconcentrations of N-Eng Ab. Representative western blots for calcineurinand α-SMA protein levels in RVFB and LVFB are shown.

FIGS. 5A-5E show that neutralizing endoglin activity reverses cardiacfibrosis after chronic right ventricular pressure overload. FIG. 5Ashows a representative histologic staining for RV collagen abundance inIgG versus N-anti-Eng Ab treated mice after moderate RVPO. FIG. 5B showsquantification of RV fibrosis after moderate RVPO is shown (n=6/group).FIGS. 5C-5E are western blots showing levels of type I collagen andcalcineurin in WT mice after moderate RVPO for 3 and 6 weeks in thepresence and absence of either an IgG control antibody or N-Eng Ab.Quantification of Type I collagen and calcineurin protein levels. *,p<0.05 vs. Sham; †, p<0.05 vs. 3 weeks RVPO, ‡, p<0.05 vs. 6 weeksRVPO+IgG.

FIG. 6 shows that reduced endoglin activity limits TGFβ1-inducedcalcineurin expression and myofibroblast transformation in rightventricular fibroblasts. (Left panel) Endoglin RV promotes fibrosis byfacilitating TGFβ1 signaling via canonical and non-canonical pathwaysincluding calcineurin-mediated myofibroblast transformation. (Rightpanel) Reduced endoglin activity in RVFB attenuates TGFβ1/calcineurinsignaling and limits myofibroblast transformation and fibrosis, therebyimproving survival.

FIGS. 7A-7F show calcineurin regulates myofibroblast transformation andTRPC-6 expression in right ventricular fibroblasts. FIG. 7A is a Westernblots showing calcineurin, α-SMA, pSmad3, total Smad3, and GAPDHexpression in human right ventricular fibroblasts (RVFB) afterstimulation with TGFβ1 (10 ng/mL for 16 to 24 hours) in the presence andabsence of cyclosporine (CS). FIGS. 7B and 7D show mRNA levels ofcalcineurin, α-SMA, and TRPC-6 in human RVFB after stimulation withTGFβ1 in the presence and absence of CS (n=3/group). FIG. 7E is aWestern blot showing silencing of TRPC-6 in human RVFB. FIG. 7F is aWestern blot showing calcineurin and α-SMA levels in human RVFB afterTGFβ1 stimulation in the presence and absence of a siRNA against TRPC-6(siTRPC-6). *P<0.05 versus vehicle; †P<0.05 versus TGFβ1 stimulation;‡P<0.05 versus WT+TGFβ1 stimulation. α-SMA indicates a-smooth muscleantigen; TGFβ1, transforming growth factor beta 1; TRPC-6, transientreceptor protein channel 6.

FIGS. 8A-8I show reduced endoglin expression limits fibrosis andcalcineurin expression in a murin model of angio-obliterative pulmonaryhypertension. FIGS. 8A-8C show RV systolic pressure, tau, and RVcompliance in Eng+/+ and Eng+/− mice after 5 weeks of treatment withSugen compound under normoxic (Su-Norm) or hypoxic (Su-Hypox) conditions(n=6/group). FIG. 8D shows mRNA levels of type I collagen in WT andEng+/− mice under Su-Norm or Su-Hypox conditions (n=6/group). FIGS. 8Eand 8F are representative histologic staining for RV collagen abundancein Eng+/+ and Eng+/− mice under Su-Norm or Su-Hypox conditions.Quantification of percent RV fibrosis is shown (n=6/group). FIG. 8Gshows mRNA levels of calcineurin, TRPC-6, and α-SMA in RV tissue from WTand Eng+/− mice under Su-Norm or Su-Hypox conditions (n=6/group).*P<0.05 versus Eng+/+Su-Norm; †P<0.05 versus Eng+/−Su-Norm; ‡P<0.05Eng+/+Su-Hypox versus Eng+/−Su-Hypox. α-SMA indicates a-smooth muscleantigen; RV, right ventricular; TRPC-6, transient receptor proteinchannel 6; WT, wild type.

FIG. 9 is a graph showing that reduced endoglin expression limitscalcineurin activity in RV pressure overload. Luciferase activity in RVlysates from Eng+/+−NFAT-Luc and Eng+/−-NFAT-Luc mice subjected to 7days of severe RVPO. *P<0.05 vs. Eng+/+−NFAT-Luc Sham; †P<0.05 vs.Eng+/+−NFAT-Luc PAC. PAC indicates pulmonary artery construction; RVPO,RV pressure overload.

FIGS. 10A-10B show RV and LV levels of TRPC1, TRPC3, TRPC4, and TRPC6 inEng+/+ and Eng+/− mice after exposure to TAC (FIG. 10A) and PAC (FIG.10B) for 10 weeks.

FIGS. 11A-11B show RV and LV levels of TRPM3, TRPM5, TRPM6, and TRPM7 inEng+/+ and Eng+/− mice after exposure to TAC (FIG. 11A) and PAC (FIG.11B) for 10 weeks.

FIGS. 12A-12B show RV and LV levels of TRPV2 and TRPV4 in Eng+/+ andEng+/− mice after exposure to TAC (FIG. 12A) and PAC (FIG. 12B) for 10weeks.

FIG. 13 is a graph showing lung type I collagen expression in Eng+/+ andEng+/− mice after exposure to TAC.

FIGS. 14A-14B are graphs showing kidney type I collagen expression andplasminogen activator inhibitor-1 (PAI-1) expression in Eng+/+ andEng+/− mice after two weeks of LV failure induced by TAC.

DETAILED DESCRIPTION

Endoglin is an important participant in the biology of right ventricle(RV) remodeling and a potential therapeutic target that modulates TGFβ1signaling, regulates calcineurin, and TRPC-6 expression. Severalfindings by the inventors, as described in detail below, have importantclinical implications. Specifically, endoglin, as a central component offibrogenic signaling in the RV, provides an important approach to reduceRV fibrosis and improve survival in RV pressure overload. Endoglin wasalso shown to be an important component in fibrogenic signaling in thelung and kidney. Further, endoglin was identified as a previouslyunrecognized positive regulator of calcineurin expression in vivo. Itwas further shown that blocking endoglin reduces RV calcineurinexpression in models of acute and chronic RV pressure overload. Inaddition, endoglin specifically regulates TGFβ1-induced calcineurinexpression and myofibroblast transformation in fibroblasts derived fromthe RV. It was also shown that endoglin regulates TRPC-6 expression inresponse to RV and LV pressure overload and that pressure overloadinduces distinct profiles of TRPC, TRPM, and TRPV expression in the RVand LV and the effects in the RV require full endoglin activity.Finally, the potential clinical utility of targeting endoglin wasexamined in mice with established RVPO by randomizing mice to aneutralizing antibody against endoglin or isotype control antibody. Inthis experiment, progressive fibrosis in the control arm and a reversalof established RV fibrosis in the anti-endoglin treatment group wasobserved. Given the importance of calcineurin/TRPC-6 in adaptive andmaladaptive cardiac remodeling, these findings implicate an importantrole for endoglin in RV remodeling and further show that targetingendoglin activity may improve RV function in heart failure, lungdisease, or kidney disease. In addition, given the importance of TGFβsignaling and its link with major profibrogenic signaling networks,these findings implicate an important role for endoglin in regulation offibrogenesis and further show that targeting endoglin activity canprovide a therapeutic approach to treating organ and tissue fibrosis.

Endoglin

Endoglin (Eng; CD105) is a 180 kDa membrane-associated dimericglycoprotein (mEng) that is also found as a circulating form composed ofthe extracellular domain, known as soluble endoglin (sEng). Endoglinplays an important role in vascular remodeling. Under basal conditionsthe vascular endothelium responds to TGFβ1 through the TGF-β type IIreceptor in association with either of two type I signaling receptorsknown as activin like kinase (ALK)1 and ALK5, which promote either aproliferative or quiescent phenotype respectively. Endoglin modulatesresponses to TGFβ1 and is implicated in the regulation of the switchfrom ALK5 to ALK1 signaling pathways. It was previously reported thatendoglin is a modulator of TGFβ1 signaling in cardiac fibroblasts andheart failure, where fibrosis plays a major role, however the role ofendoglin in cardiac remodeling, specifically in the right ventricle hasbeen largely unexplored.

Right Ventricle Cardiac Remodeling, TGFβ1 Signaling, Endoglin,Calcineurin, and TRP Signaling

Previous studies of TGFβ1 activity in cardiac remodeling have been morefocused on left ventricular failure. It was recently reported thatreduced endoglin activity limits LV fibrosis by attenuating canonicaland non-canonical TGFβ1 signaling in a murine model of left heartfailure. In those studies, the effect of reduced endoglin activity on LVcalcineurin expression was not observed. Several other studies haveshown that both TGFβ1 and calcineurin play critical roles in regulatingLV responses to injury (Kuwahara et al., Circulation. 106:130-135, 2002,Kapur et al., Circulation. 125:2728-2738, 2012, White et al., A. TherAdv Cardiovasc Dis. 6:5-14, 2012, Fickenberg et al., Am J Pathol.163:355-366, 2003, Davis et al., Dev Cell. 23:705-715, 2012, Heineke etal., J Mol Cell Cardiol. 48:1080-1087, 2010, Berry et al., Circ Res.109:407-417, 2011); however, no studies have examined a functionalinteraction between TGFβ1 and calcineurin in RV remodeling. Here, amouse model of pulmonary artery constriction was used to uncouple the RVfrom the pulmonary vasculature and to explore the direct impact ofpressure overload on RV remodeling. It was first observed that RVendoglin expression is increased in response to RVPO and then it wasshown that endoglin promotes RV fibrosis by facilitating TGFβ1 signalingthrough canonical and non-canonical pathways.

In both in vivo and in vitro studies, a neutralizing antibody toendoglin (N-Eng Ab, TRC105), which is an IgG1 antibody that binds bothhuman and mouse endoglin with high avidity was used. TRC105 has beenstudied extensively in cancer biology and is known to bind and disruptendoglin signaling in endothelium (Rosen et al., Clin Cancer Res.18:4820-4829, 2012, Seon et al., Curr Drug Deliv. 8:135-143, 2011). Ithas been shown that TRC105 blocks endoglin activity in cardiacfibroblasts. To begin exploring the potential clinical utility ofblocking endoglin as a treatment for adverse RV remodeling, a randomizedstudy in WT mice subjected to moderate RVPO for 3 weeks then treatedwith either TRC105 or an isotype control IgG Ab for an additional 3weeks was performed. After 6 weeks, progressive RV fibrosis in thecontrol arm and reduced RV fibrosis in the anti-endoglin treated groupwas observed. Collectively, these findings confirm that targetingendoglin using an antibody mediated approach can prevent the developmentof RV fibrosis in acute RVPO and reverse established RV fibrosis in achronic model of moderate RVPO.

To further explore the dependence of TGFβ1-induced calcineurinexpression and myofibroblast transformation on endoglin, cardiacfibroblasts were studied in vitro. Using WT and Eng+/− mice, it wasfirst identified that endoglin was required for TGFβ1-inducedcalcineurin expression and myofibroblast transformation in RV, but notLV fibroblasts. This observation was confirmed by blocking endoglin withthe N-Eng Ab, TRC105, which also attenuated TGFβ1-induced calcineurinexpression and myofibroblast transformation in RV, not LV fibroblasts.In both loss-of-function studies, it was observed that reducing endoglinactivity limited phosphorylation of Smad-3 and ERK-1/2 in both RV and LVfibroblasts, thereby attenuating expression of type I collagen,suggesting that endoglin plays an important role in regulatingbiventricular TGFβ1 signaling with a potentially unique role forendoglin in the TGFβ1/calcineurin pathway that is specific tofibroblasts of RV origin.

Transient receptor potential (TRP) channels of multiple subclasses areexpressed in the heart, including cardiomyocytes, fibroblasts,endothelial cells, and vascular smooth muscle cells (Nilius et al.,Physiol Rev. 87:165-217, 2007; Watanabe et al., Pharmacol Ther.118:337-351, 2008). TRP channels expressed in the heart most likelycoordinate signaling within local domains or through direct interactionwith Ca²⁺-dependent regulatory proteins (Eder et al., Circ Res.108:265-272, 2011). The TRPC subclass appears to regulate the cardiachypertrophic response. In particular, TRPC3 and TRPC6 were implicated inangiotensin II-induced nuclear factor of activated T-cells (NFAT)activation in isolated cardiomyocytes (Onohara et al., EMBO J.25:5305-5316, 2006), which is an essential step of cardiac hypertrophydevelopment in the whole heart. The TRPM subclass, particularly TRPM4,has been proposed to generate a Ca²⁺-activated nonselective Ca²⁺ channel(NSCC) in atrial myocytes that might be responsible for delayedafterdepolarizations (Guinamard et al., J Physiol. 558:75-83, 2004).Several TRPs have also been implicated in blood pressure regulation,among those are the TRPM4, TRPV1, TRPV4, TRPC1, and TRPC6 channels(Dietrich et al., Thromb Haemost. 103:262-270, 2005; Mathar et al., JClin. Invest. 120:3267-3279, 2010; Willette et al., J Pharmacol ExpTher. 326, 443-452, 2008; Pacher et al., J Physiol. 558:647-657, 2004;Suzuki et al., J Biol Chem 278.22664-22668, 2003).

To explore a functional role for endoglin as a regulator of TRPM, TRPV,and TRPC expression in response to RV or LV pressure overload,Eng+/−(endoglin haploinsufficient) and Eng+/+(wild-type) mice wereexposed to thoracic aortic (TAC) or pulmonary arterial (PAC)construction for 10 weeks. Analysis of biventricular tissue by real-timepolymerase chain reaction (RT-PCR) showed that pressure overload induceddistinct profiles of TRPM, TRPV, and TRPC expression in the RV and LV ofmice and the effects, particularly in the RV, require full endoglinactivity. It was further shown that endoglin is necessary for TGFβ1induced increase in expression of TRPC-6 and α-SMA by acalcineurin-dependent mechanism in human RV fibroblasts and that TRPC-6mediates a feedback loop promoting calcineurin expression andmyofibroblast transformation in human RV fibroblasts that is alsodependent on endoglin. In Eng+/− mice exposed to Sugen+ hypoxia, reducedendoglin activity improved RV diastolic function, limited fibrosis, andattenuated expression of calcineurin, TRPC-6, and α-SMA. Taken together,the data support that endoglin is also an important regulator of TRPexpression in modulating RV responses to injury.

TGFβ Signaling and Fibrosis

TGFβ belongs to Th1 cytokines and is synthesized by a wide variety ofcells including macrophages, mononuclear cells, and fibroblasts. TGFβ1,TGFβ2, TGFβ3 form the TGFβ subfamily and their synthesis is cell type-and context-dependent with unique as well as similar functions. TGFβ isa major player in initiation and progression of fibrogenesis. Inresponse to vascular injury, infiltrated mononuclear cells produce TGFβand other growth factors in the wound area. As a chemo-attractant, TGFβattracts neutrophils to the wound site and thus acts as an inflammatorycytokine in the initial stage of wound healing. TGFβ also inducesmigration of fibroblasts from the vicinity of wounds, and fibroblast tomyofibroblasts differentiation. TGFβ-activated fibroblasts ordifferentiated myofibroblasts are the major cell-type that synthesizescollagen and other extra-cellular matrix proteins to heal the damagedtissues. Specifically, TGFβ1-induced phosphorylation of Smads-2/3 andERK promotes Type I collagen synthesis and fibroblast proliferation.However, sustained activation of myofibroblasts, due to chronicinflammation and TGFβ signaling, leads to the development of fibrosisand eventually organ failure.

Given that the inventors previously reported that reduced endoglinactivity limits LV fibrosis by attenuating canonical and non-canonicalTGFβ1 signaling in a murine model of left heart failure it is an objectof the present invention to investigate the role of endoglin inmodulating fibrotic signaling via TGFβ1 signaling in other organs. Toexamine whether fibrotic signaling in other organ tissues isendoglin-dependent, type 1 collagen expression was analyzed in lungtissue and kidney tissue of Eng+/+ and Eng+/− mice. The results showthat reduced endoglin expression attenuates increased collagenexpression in lungs and kidneys, thus, indicating that endoglin isrequired for regulation of fibrotic signaling and modulation of endoglinactivity would be useful in the context of tissue fibrosis.

Soluble Endoglin

The methods of the invention can, in certain embodiments, employ solubleendoglin, a soluble endoglin fragment, or a soluble endoglin analog,e.g., a fragment or an analog that retains the ability to bind TGFβ1.

Full length endoglin is a 180 kDa homodimeric co-receptor for members ofthe TGF-β superfamily. Two isoforms of endoglin are known: a 633 aminoacid protein and 600 amino acid protein. These two forms differ in thelength of their cytoplasmic tail; the longer form has 47 amino acid tail(L-mEng), whereas the shorter form has a 14 amino acid cytoplasmic tail(S-mEng). The amino acid sequences of endoglin are described in NCBIaccession numbers NP_001108225 and NP_000109.1 and are shown in FIG. 10.The mature endoglin sequences include amino acids 26 to 658 of isoform 1and amino acids 26-625 of isoform 2. In both isoforms, amino acids 587to 611 are predicted to be the transmembrane domain. The correspondingextracellular region (amino acids 26 to 586 or 27 to 586) of endoglin,fragments thereof, or analogs thereof may therefore be used in theinvention.

The methods described herein can also use a fragment of soluble endoglin(e.g., any of those described herein. Preferred fragments are capable ofbinding TGFβ1, e.g., with at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%,75%, 80%, 85%, 90%, 95%, 97%, or 99% of the binding affinity of solubleendoglin or the naturally occurring form of soluble endoglin.

The methods described herein can also use a soluble endoglin analog. Incertain embodiments, the analog has at least 60%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% sequence identity to soluble endoglin or to asoluble endoglin fragment. Preferred analogs are capable of bindingTGFβ1, e.g., with at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%,80%, 85%, 90%, 95%, 97%, or 99% of the binding affinity of solubleendoglin.

Antibodies

The methods of the invention can employ an antibody that preventsendoglin activity or an antigen-binding fragment thereof. In certainembodiments, the antibody specifically binds to mEng or to sEng. Theantibody can bind specifically to the extracellular domain (ECD) ofmEng, the residual membrane-associated component of mEng after cleavageof the ECD, or to circulating sEng. The antibody can be a monoclonal ora polyclonal antibody. In certain embodiments, the antibody ishumanized. The antibody or antibody fragment can be a single chainantibody (scFv), Fab, Fab′2, scFv, SMIP, diabody, nanobody, aptamer, ordomain antibody.

Antibodies (e.g., monoclonal, polyclonal, poly-specific, ormono-specific antibodies) against endoglin (e.g., antagonisticantibodies) can be made using any of the numerous methods for makingantibodies known in the art. In one example, the relevant endoglinsequence is produced as a C-terminal fusion with glutathioneS-transferase (GST) (Smith et al., Gene 67:31-40, 1988). The fusionprotein is purified on glutathione-Sepharose beads, eluted withglutathione, cleaved with thrombin (at an engineered cleavage site), andpurified for immunization of rabbits. Primary immunizations are carriedout with Freund's complete adjuvant and subsequent immunizations withFreund's incomplete adjuvant. Antibody titers are monitored by Westernblot and immunoprecipitation analyses using the thrombin-cleaved proteinfragment of the GST fusion protein. Immune sera are affinity purifiedusing CNBr-Sepharose-coupled protein. Antiserum specificity can bedetermined using a panel of unrelated GST proteins.

Alternatively, monoclonal antibodies that specifically bind endoglin canbe prepared using standard hybridoma technology (see, e.g., Kohler etal., Nature 256:495-7, 1975; Kohler et al., Eur. J. Immunol. 6:511-9,1976; Kohler et al., Eur. J. Immunol. 6:292-5, 1976; Hammerling et al.,Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981). Onceproduced, monoclonal antibodies can also be tested for specificrecognition by Western blot or immunoprecipitation analysis.Alternatively, monoclonal antibodies can be prepared using thepolypeptide of the invention described above and a phage display library(Vaughan et al., Nat. Biotechnol. 14:309-14, 1996).

In order to generate polyclonal antibodies on a large scale and at a lowcost an appropriate animal species can be chosen. Polyclonal antibodiescan be isolated from the milk or colostrum of, e.g., immunized cows.Bovine colostrum contains 28 g of IgG per liter, while bovine milkcontains 1.5 g of IgG per liter (Ontsouka et al., J. Dairy Sci.86:2005-11, 2003). Polyclonal antibodies can also be isolated from theyolk of eggs from immunized chickens (Sarker et al., J. Pediatr.Gastroenterol. Nutr. 32:19-25, 2001).

Useful antibodies can be identified in several different screeningassays. First, antibodies are assayed by ELISA to determine whether theyare specific for the immunizing antigen (i.e., endoglin).

Using standard techniques, ELISA plates are coated with immunogen, theantibody is added to the plate, washed, and the presence of boundantibody detected by using a second antibody specific for the Ig of thespecies in which the antibody was generated.

RNA Interference

The methods described herein can also use RNAi to inhibit endoglinexpression. RNA interference (RNAi) is a mechanism ofpost-transcriptional gene silencing (PTGS) in which double-stranded RNA(dsRNA) corresponding to a gene or mRNA of interest is introduced intoan organism, resulting in the degradation of the corresponding mRNA. Inthe RNAi reaction, both the sense and anti-sense strands of a dsRNAmolecule are processed into small RNA fragments or segments ranging inlength from 21 to 23 nucleotides (nt) and having 2-nucleotide 3′ tails.Alternatively, synthetic dsRNAs, which are 21 to 23 nt in length andhave 2-nucleotide 3′ tails, can be synthesized, purified, and used inthe reaction. These 21 to 23 nt dsRNAs are known as “guide RNAs” or“short interfering RNAs” (siRNAs).

The siRNA duplexes then bind to a nuclease complex composed of proteinsthat target and destroy endogenous mRNAs having homology to the siRNAwithin the complex. The complex functions by targeting the homologousmRNA molecule through base pairing interactions between one of the siRNAstrands and the endogenous mRNA. The mRNA is then cleaved approximately12 nt from the 3′ terminus of the siRNA and degraded. In this manner,specific genes can be targeted and degraded, thereby resulting in a lossof protein expression from the targeted gene. siRNAs can also bechemically synthesized or obtained from a company that chemicallysynthesizes siRNAs (e.g., Dharmacon Research Inc., Pharmacia, or ABI).Endoglin RNAi molecules are commercially available and can be obtainedfrom a variety of sources, including Santa Cruz Biotechnology (siRNA;Cat. No. sc-35302). The specific requirements and modifications of dsRNAare described in PCT Publication No. WO 01/75164, and in U.S. PatentApplication Publication No. 20060067937 and PCT Publication No. WO06/034507, incorporated herein by reference.

Small Molecule Inhibitors

Small molecule inhibitors of endoglin activity can be screened for usingmethods known in the art. High-throughput screening techniques can beused to identify candidate small molecules that modulate, alter, ordecrease endoglin expression or biological activity (e.g., a decrease byat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or morecompared to a normal reference).

In particular examples, candidate small molecules having one or more ofthe following properties are considered inhibitors of endoglin activity:decrease endoglin expression, reduced TGFβ1 signaling, reducedphosphorylated Smad 2/3 and mitogen activated protein kinases (e.g.,ERK), reduced calcineurin expression, or reduced reactive oxygen species(ROS) production, compared to a control or a normal reference. Candidatesmall molecules can be tested for their effect on endoglin activityusing assays known in the art.

Candidate small molecules can also be tested for their effect onendoglin activity using any particular cell based assays describedherein. Standard methods may be used to measure analyte levels orcellular parameters in any bodily fluid, including, but not limited to,urine, blood, serum, plasma, saliva, or cerebrospinal fluid. Suchmethods include immunoassay, ELISA, Western blotting using antibodiesdirected to endoglin and quantitative enzyme immunoassay techniques.ELISA assays are the preferred method for measuring polypeptide levels.Accordingly, the measurement of antibodies specific to endoglin in asubject may also be used to determine if a compound has effects onendoglin activity.

In one embodiment, a compound that affects endoglin activity may show adecrease in the expression of a nucleic acid encoding endoglin. Methodsfor detecting such alterations are standard in the art. In one exampleNorthern blotting or real-time PCR is used to detect mRNA levels.

In another embodiment, hybridization techniques may be used to monitorexpression levels of a gene encoding a polypeptide of the invention upontreatment with a candidate compound.

In a further embodiment, a reporter gene such as a gene encoding GFP orluciferase can be fused to the endoglin promoter to monitor theexpression levels of endoglin upon treatment with a candidate compound.

In general, candidate compounds are identified from large libraries ofboth natural product or synthetic (or semi-synthetic) extracts, chemicallibraries, according to methods known in the art. Those skilled in thefield of drug discovery and development will understand that the precisesource of test extracts or compounds is not critical to the screeningprocedure(s) of the invention.

Proteases

The compositions of the invention can include proteases, particularlymatrix metalloproteinase 14 (MMP-14). MMP-14 (UniProtKB:P50281) is aknown cleavage protease of the endoglin receptor. The advantages ofprotease cleavage of the endoglin receptor is that 1) cleavage of theendoglin receptor can be a companion diagnostic with soluble endoglin tomeasure the efficacy and identify optimal candidates for anti-endoglintherapy and 2) the release of soluble endoglin as a result of proteasecleavage would provide feedback to further inhibit endoglin signalingthereby enhancing potency of the compositions described herein. In someembodiments, the composition can include a polypeptide having an aminoacid sequence having at least 80% identity (e.g., at least 85%, 90%,92%, 95%, 96%, 97%, 98%, or 99%) to the amino acid sequence of MMP-14shown below and having protease activity.

(SEQ ID NO: 1) MSPAPRPPRCLLLPLLTLGTALASLGSAQSSSFSPEAWLQQYGYLPPGDLRTHTQRSPQSLSAAIAAMQKFYGLQVTGKADADTMKAMRRPRCGVPDKFGAEIKANVRRKRYAIQGLKWQHNEITFCIQNYTPKVGEYATYEAIRKAFRVWESATPLRFREVPYAYIREGHEKQADIMIFFAEGFHGDSTPFDGEGGFLAHAYFPGPNIGGDTHFDSAEPWTVRNEDLNGNDIFLVAVHELGHALGLEHSSDPSAIMAPFYQWMDTENFVLPDDDRRGIQQLYGGESGFPTKMPPQPRTTSRPSVPDKPKNPTYGPNICDGNFDTVAMLRGEMFVFKERWFWRVRNNQVMDGYPMPIGQFWRGLPASINTAYERKDGKFVFFKGDKHWVFDEASLEPGYPKHIKELGRGLPTDKIDAALFWMPNGKTYFFRGNKYYRFNEELRAVDSEYPKNIKVWEGIPESPRGSFMGSDEVFTYFYKGNKYWKFNNQKLKVEPGYPKSALRDWMGCPSGGRPDEGTEEETEVIIIEVDEEGGGAVSAAAVVLPVLLLLLVLAVGLAVFFFRRHGTPRRLLYCQRSLLDKVMMP-14 belongs to a class of matrix metalloproteinases (MMPs) within thesuper family of zinc endopeptidases. The protease contains sevendomains: a signal peptide leading MMP-14 into the secretory pathway, apropeptide domain maintaining MMP in a latent form, a catalytic domainresponsible for enzymatic activity, a hinge region maintaining properconformation, a hemopexin domain required for substrate reorganization,a transmembrane domain anchoring MMP into the plasma membrane, and acytoplasmic domain required for endocytosis (Stocker et al., Curr OpinStruct Biol. 3:383-390, 1995, Knauper et al., J Biol Chem.271:17124-17131, 1996).

The catalytic domain, or active fragment of MMP-14, is a highlyconserved motif containing a methionine and three histidines that bind azinc ion in the catalytic site. In some embodiments, the compositionincludes an active fragment of MMP-14, for example, an active fragmenthaving at least 90% (e.g., at least 92%, 95%, 96%, 97%, 98%, or 99%)identity to the amino acid sequence below.

(SEQ ID NO: 2) AIQGLKWQHNEITFCIQNYTPKVGEYATYEAIRKAFRVWESATPLRFREVPYAYIREGHEKQADIMIFFAEGFHGDSTPFDGEGGFLAHAYFPGPNIGGDTHFDSAEPWTVRNEDLNGNDIFLVAVHELGHALGLEHSSDPSAIMAPFYQWMDTENFVLPDDDRRGIQQLYGGESGConditionsChemotherapy and Radiation Therapy-Induced Cardiotoxicity

The observations that endoglin is a regulator of calcineurin expressionin the RV and can serve as a novel therapeutic target to limit fibrosisand improve survival in RV pressure overload have important implicationsfor RV failure in multiple clinical settings.

Anticancer therapies (e.g., chemotherapy) and radiation therapies haveled to a long life expectancy for many patients; howevertreatment-related cardiac toxicity can be a side effect of anticancertherapies and radiation therapies that increases the mortality rate inthese patients. The compositions of the invention, therefore, can beadministered prior to or concurrently with the start of anticancertherapies or radiation therapies to provide cardioprotection and reducethe incidence of cardiac toxicity. Additionally, the compositions can beadministered following the development of chemotherapy or radiationinduced heart disease or heart failure. Furthermore, the dosing andtiming for administration of the composition of the invention depends ondifferent factors related to the type of chemotherapeutic agent, doseadministered during each cycle, cumulative dose, schedule ofadministration, route of administration, combination of othercardiotoxic drugs or association with radiotherapy, age of the subject,presence of cardiovascular risk factors, or previous cardiovasculardisease.

The composition can be administered in a therapeutically effectiveamount prior to the start of chemotherapy or radiation therapy (e.g., 4weeks prior, 3 weeks prior, 2 weeks prior, 1 week prior, 6 days prior, 5days prior, 4 days prior, 3 days prior, 2 days prior, 1 day prior,within less than 24 hours prior to the start of chemotherapy orradiation therapy). The administration of the composition of theinvention can be continued throughout the duration of chemotherapy orradiation therapy and extends past the conclusion of chemotherapy orradiation therapy (e.g., extended 1 day, 2, days, 3 days, 4 days, 5days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks after theconclusion of chemotherapy or radiation therapy). The composition canalso be administered concurrently with the start of chemotherapy orradiation therapy (e.g., before 24 hours after the start ofchemotherapy, administered daily, twice daily, every other day, everyother week, and in doses of less than about 3 mg/kg (e.g., 2.9 mg/kg,2.8 mg/kg, 2.7 mg/kg, 2.6 mg/kg, 2.5 mg/kg, 2.3 mg/kg, 2.2 mg/kg, 2.1mg/kg, 2.0 mg/kg, 1.8 mg/kg, 1.7 mg/kg, 1.5 mg/kg, 1.2 mg/kg, 0.5 mg/kg,0.3 mg/kg), or more than about 3.5 mg/kg (e.g., 3.6 mg/kg, 3.8 mg/kg,4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7mg/kg, 10 mg/kg, 15 mg/kg).

A reduction in cardiac damage can be quantitatively measured by animprovement in a cardiovascular parameter (e.g., end-diastolic volume(EDV), end-systolic volume (ESV), stroke volume, ejection fraction,heart rate, and cardiac output) when compared to normal ranges (e.g., anend-diastolic volume (EDV) from about 65-240 mL, an end-systolic volume(ESV) from about 16-143 mL, a stroke volume from about 55-100 mL, anejection fraction from about 55-70%, a heart rate from about 60-100 bpm,and/or cardiac output of about 4.0-8.0 L/min).

Autoimmune Disease, Non-Autoimmune Inflammatory Diseases, OrganTransplantation

A previously unrecognized functional role for endoglin as a regulator ofcalcineurin signaling and myofibroblast transformation was observed.Using Eng+/− mice and WT mice treated with a neutralizing antibodyagainst endoglin, an improved survival and a significant reduction in RVfibrosis compared to WT controls after 7 days of severe RVPO wasobserved. These findings confirmed an important role for endoglin in RVfibrosis; however the most dramatic observation was the complete loss ofcalcineurin expression in the pressure-overloaded RV and associatedreduction in levels of genes upregulated by calcineurin, including MYH7and TRPC-6. Consistent with the report from Davis et al. (Dev Cell.23:705-715, 2012) implicating an important role for calcineurin/TRPC-6as regulators of myofibroblast transformation, an association betweenreduced TRPC-6 and α-SMA levels in the RV was observed, suggesting adisruption of myofibroblast transformation despite increased tissuelevels of active TGFβ1. These data identify endoglin as an essentialcomponent of RV remodeling and a potential therapeutic target thatregulates calcineurin expression, reduces RV fibrosis, and improvessurvival in RV pressure overload.

The compositions of the invention can be used alone or in combinationwith inhibitors of the calcineurin pathway to treat autoimmune disease,non-autoimmune inflammatory disease, and/or organ transplantation.Examples of autoimmune disease and inflammatory diseases include, butare not limited to acne vulgaris, asthma, autoimmune diseases (e.g.,acute disseminated encephalomyelitis (ADEM), Addison's disease,agammaglbulinemia, alopecia areata, amyotrophic lateral sclerosis,ankylosing spondylitis, antiphospholipid syndrome, antisynthetasesyndrome, atopic allergy, atopic dermatitis, autoimmune aplastic anemia,autoimmune cardiomyopathy, autoimmune enteropathy, autoimmunehemolyticanemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmunelymphoproliferative syndrome, autoimmune peripheral neuropathy,autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmuneprogesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmuneurticaria, autoimmune uveitis, Balo concentric sclerosis, Behcet'sdisease, Berger's disease, Bickerstaff's encephalitis, Blau syndrome,bullous pemphigoid, Castleman's disease, celiac disease, Chagas disease,chronic inflammatory demyelinating polyneuropathy, chronic recurrentmultifocal osteomyelitis, chronic obstructive pulmonary disease,Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, coldagglutinin disease, complement component 2 deficiency, contactdermatitis, cranial arteritis, CREST syndrome, Crohn's disease,Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Dego'sdisease, Dercum's disease, dermatitis herpetiformis, dermatomyositis,diabetes mellitus type 1, diffuse cutaneous systemic sclerosis,Dressler's syndrome, drug-induced lupus, discoid lupus erythematosus,eczema, endometriosis, enthesitis-related arthritis, eosinophilicfasciitis, eosinophilic gastroenteritis, epidermolysis bullosaacquisita, erythema nodosum, erythroblastosis fetalis, essential mixedcryoglobulinemia, Evan's syndrome, fibrodysplasia ossificansprogressive, fibrosing alveolitis, gastritis, gastrointestinalpemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture'ssyndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto'sencephalopathy, Hashimoto's thyroiditis, Henoch-Schonlein purpura,herpes gestationis, hidradenitis suppurativa, Hughes-Stovin syndrome,hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases,idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion bodymyositis, chronic inflammatory demyelinating polyneuropathy,interstitial cystitis, juvenile idiopathic arthritis, Kawasaki'sdisease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis,lichen planus, lichen sclerosus, linear IgA disease, lupuserythematosus, Majeed syndrome, Meniere's disease, microscopicpolyangiitis, mixed connective tissue disease, morphea, Mucha-Habermanndisease, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica,neuromyotonia, ocular cicatricial pemphigoid, opsoclonus myoclonussyndrome, Ord's thyroiditis, palindromic rheumatism, PANDAS,paraneoplastic cerebellar degeneration, paroxysmal nocturnalhemoglobinuria, Parry Romberg syndrome, Parsonage-Turner syndrome, parsplanitis, pemphigus vulgaris, pernicious anaemia, perivenousencephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgiarheumatic, polymyositis, primary biliary cirrhosis, primary sclerosingcholangitis, progressive inflammatory neuropathy, psoriatic arthritis,psoriasis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen'sencephalitis, raynaud phenomenon, relapsing polychondritis, Reiter'ssyndrome, restless leg syndrome, retroperitoneal fibrosis, rheumaticfever, Schnitzler syndrome, scleritis, scleroderma, serum sickness,Sjogren's syndrome, spondyloarthropathy, stiff person syndrome, subacutebacterial endocarditis, Susac's syndrome, Sweet's syndrome, sympatheticophthalmia, Takayasu's arteritis, temporal arteritis, thrombocytopenia,Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis,undifferentiated connective tissue disease, undifferentiatedspondyloarthropathy, vitiligo, and Wegener's granulomatosis), celiacdisease, chronic prostatitis, glomerulonephritis, hypersensitivities,inflammatory bowel diseases, pelvic inflammatory disease, reperfusioninjury, sarcoidosis, transplant rejection, vasculitis, interstitialcystitis, and osteoarthritis.

The compositions of the invention are also expected to be effective intreating ischemia-reperfusion injury from reconstructive and organtransplantation procedures. Exemplary tissues and organs to be treatedusing the composition of the invention have active metabolism andincreased mitochondrial function and are susceptible to reperfusioninjury after brief periods of ischemia and include but are not limitedto; skeletal muscle, the heart, the liver, large intestine, smallintestine, the brain, the skin, the limbs (e.g., arms, legs, feet,hands).

Conditions Associated with Oxidative Stress

Reports have identified that TGFβ1 activates calcineurin expression andactivity by generating reactive oxygen species (ROS), thus, impairedfunction of the TGFβ1 co-receptor, endoglin, should limit calcineurinexpression and activity by reducing ROS. Accordingly, the composition ofthe invention can be used to treat conditions associated with oxidativestress related to increase ROS production. Examples of conditionsassociated with oxidative stress include, but are not limited toreperfusion injury, wound healing, toxic hepatitis, viral hepatitis,chronic organ disease (e.g., chronic lung disease, chronic obstructivepulmonary disease, chronic viral hepatitis, chronic renal disease,chronic pancreatitis, chronic prostatitis, chronic inherited bleedingdisorders (e.g., hemophilia, von Willebrand disease), and chronic bonedisease (e.g., osteogenesis imperfect, Paget's disease), oxidativestress from dialysis, renal toxicity, kidney failure, ulcerativecolitis, bacterial infection, viral infections, upper respiratory tractdiseases, oxidative stress due to sun damage, eczema, atopic dermatitis,polymyositis, and dermatitis herpetiformis.

Other conditions that may be treated using the compositions of theinvention include cancers. Cancers are generally characterized byunregulated cell growth, formation of malignant tumors, and invasion tonearby parts of the body. Cancers may also spread to more distant partsof the body through the lymphatic system or bloodstream. Cancers may bea result of gene damage due to tobacco use, certain infections,radiation, lack of physical activity, obesity, and/or environmentalpollutants. Cancers may also be a result of existing genetic faultswithin cells to cause diseases due to genetic heredity. Screenings maybe used to detect cancers before any noticeable symptoms appear andtreatment may be given to those who are at higher risks of developingcancers (e.g., people with a family history of cancers). Examples ofscreening techniques for cancer include but are not limited to physicalexamination, blood or urine tests, medical imaging, and/or genetictesting. Non-limiting examples of cancers include: bladder cancer,breast cancer, colon and rectal cancer, endometrial cancer, kidney orrenal cell cancer, leukemia, lung cancer, melanoma, Non-Hodgkinlymphoma, pancreatic cancer, prostate cancer, ovarian cancer, stomachcancer, wasting disease, and thyroid cancer.

Fibrotic Diseases

Fibrotic disease represents one of the largest groups of disorders forwhich there is no effective therapy. Fibrotic diseases are characterizedby excessive scarring due to excessive production, deposition, andcontraction of extracellular matrix. This process usually occurs overmany months and years, and can lead to organ dysfunction or death.Examples of fibrotic diseases include, but are not limited to,idiopathic pulmonary fibrosis, organ fibrosis, interstitial lungdisease, skin fibrosis, diabetic nephropathy, liver fibrosis, livercirrhosis, nonalcoholic steatohepatitis (NASH), rheumatoid arthritis,fibrosarcomas, keloids and hypertrophic scars, arteriosclerosis, kidneydisease, macular degeneration, retinal and vitreal retinopathy, surgicalcomplications, chemotherapeutic drug-induced fibrosis, radiation-inducedfibrosis, accidental injury, burns, local scleroderma, and systemicscleroderma. Rheumatoid arthritis and other connective tissue disordersoften have associated lung pathologies. Lung fibrosis alone can be amajor cause of death in scleroderma lung disease, idiopathic pulmonaryfibrosis, radiation- and chemotherapy-induced lung fibrosis and inconditions caused by occupational inhalation of dust particles.

Tissue fibrosis is generally considered to arise due to a failure of thenormal wound healing response to terminate. After injury, new connectivetissue needs to be synthesized. During this process, mesenchymalfibroblasts become “activated” in that they proliferate and migrate intothe wound and synthesize elevated levels of matrix proteins, includingcollagen and fibronectin. The mesenchymal cells activated during tissuerepair and wound healing in kidney and liver are called mesangial cellsand stellate cells, respectively. The fibroblasts present in a wound area specialized form of fibroblasts termed myofibroblasts as they expresselevated levels of α-SMA and consequently display a markedly enhancedability to contract extracellular matrix. This aspect of fibroblastfunction is necessary for wound closure. Myofibroblasts are present inabundance within fibrotic lesions and thus contribute to the excessivescarring observed in lesions of fibrotic disease. Myofibroblasts infibrotic tissues are derived from at least three sources: expansion andactivation of resident tissue fibroblasts, transition of epithelialcells into mesenchymal cells (epithelial-mesenchymal transition, EMT),and tissue migration of bone marrow-derived circulating fibrocytes.Endothelial to mesenchymal transition (EndoMT) is another possiblesource of tissue myofibroblasts. EndoMT is a biological process in whichendothelial cells lose their specific markers and acquire a mesenchymalor myofibroblastic phenotype and express mesenchymal cell products suchas α-SMA and type I collagen. Similar to EMT, EndoMT can be induced byTGFβ.

Reduced endoglin expression is shown to attenuate increased collagenexpression in lungs and kidneys subjected to increased venous pressureand decreased perfusion and to limit fibrosis in the RV and/or LV inmodels of heart failure and pulmonary hypertension. Thus, it isenvisioned that the compositions of the invention can be used to treatfibrotic diseases (e.g., organ fibrosis) where endoglin plays a role inmodulating fibrotic signaling.

Administration and Dosage

The methods described herein feature administration of a compositionthat inhibits endoglin activity. The composition can be formulated foruse in a variety of drug delivery systems. One or more physiologicallyacceptable excipients or carriers can also be included in thecomposition for proper formulation. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MackPublishing Company, Philadelphia, Pa., 17th ed., 1985. For a briefreview of methods for drug delivery, see, e.g., Langer (Science249:1527-1533, 1990).

The pharmaceutical composition can be used for parenteral, intranasal,topical, oral, or local administration, such as by a transdermal means,for prophylactic and/or therapeutic treatment. The pharmaceuticalcomposition can be administered parenterally (e.g., by intravenous,intramuscular, or subcutaneous injection), or by oral ingestion, or bytopical application or intraarticular injection at areas affected by thevascular or cancer condition. Additional routes of administrationinclude intravascular, intra-arterial, intratumor, intraperitoneal,intraventricular, intraepidural, as well as nasal, ophthalmic,intrascleral, intraorbital, rectal, topical, or aerosol inhalationadministration. Sustained release administration is also specificallyincluded in the invention, by such means as depot injections or erodibleimplants or components. Thus, the invention provides compositions forparenteral administration that include the above mention agentsdissolved or suspended in an acceptable carrier, preferably an aqueouscarrier, e.g., water, buffered water, saline, PBS, and the like. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents, detergents and the like. The invention also providescompositions for oral delivery, which may contain inert ingredients suchas binders or fillers for the formulation of a tablet, a capsule, andthe like. Furthermore, this invention provides compositions for localadministration, which may contain inert ingredients such as solvents oremulsifiers for the formulation of a cream, an ointment, and the like.

These compositions may be sterilized by conventional sterilizationtechniques or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably between 5 and 9 or between 6 and 8, and mostpreferably between 7 and 8, such as 7 to 7.5. The resulting compositionsin solid form may be packaged in multiple single dose units, eachcontaining a fixed amount of the above-mentioned agent or agents, suchas in a sealed package of tablets or capsules. The composition in solidform can also be packaged in a container for a flexible quantity, suchas in a squeezable tube designed for a topically applicable cream orointment.

The compositions containing an effective amount can be administered forprophylactic or therapeutic treatments. In prophylactic applications,compositions can be administered to a subject diagnosed as being at riskfor heart failure (e.g., having lower levels of soluble endoglin, asdescribed in U.S. patent application Ser. No. 13/288,493). Compositionsof the invention can be administered to the subject (e.g., a human) inan amount sufficient to delay, reduce, or preferably prevent the onsetof the disorder. In therapeutic applications, compositions areadministered to a subject (e.g., a human) already suffering from heartfailure of any of the disorders described herein in an amount sufficientto cure or at least partially arrest the symptoms of the disorder andits complications. An amount adequate to accomplish this purpose isdefined as a “therapeutically effective amount,” an amount of a compoundsufficient to substantially improve at least one symptom associated withthe disease or a medical condition. For example, in the treatment ofheart failure, an agent or compound that decreases, delays, suppresses,or arrests any symptom of the condition would be therapeuticallyeffective. A therapeutically effective amount of an agent or compound isnot required to cure a disease or condition but will provide a treatmentfor a disease or condition such that the onset of the disease orcondition is delayed, hindered, or prevented, or the disease orcondition symptoms are ameliorated, or the term of the disease orcondition is changed or, for example, is less severe or recovery isaccelerated in an individual.

Amounts effective for this use may depend on the severity of the diseaseor condition and the weight and general state of the subject. Thetherapeutically effective amount of the compositions of the inventionand used in the methods of this invention applied to mammals (e.g.,humans) can be determined by the treating physician with considerationof individual differences in age, weight, and the condition of themammal. The agents of the invention are administered to a subject (e.g.a mammal, such as a human) in an effective amount, which is an amountthat produces a desirable result in a treated subject (e.g., reductionof cardiac fibrosis). Therapeutically effective amounts can also bedetermined empirically by those of skill in the art.

Single or multiple administrations of the compositions of the inventionincluding an effective amount can be carried out with dose levels andpattern being selected by the treating physician. The dose andadministration schedule can be determined and adjusted based on theseverity of the disease or condition in the subject, which may bemonitored throughout the course of treatment according to the methodscommonly practiced by clinicians or those described herein.

The compounds of the present invention may be used in combination witheither conventional methods of treatment or therapy or may be usedseparately from conventional methods of treatment or therapy.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to an individual. Alternatively, pharmaceuticalcompositions according to the present invention may be comprised of acombination of a compound of the present invention in association with apharmaceutically acceptable excipient, as described herein, and anothertherapeutic or prophylactic agent known in the art.

Combination Therapy

Anticancer/Anti-Proliferative Drugs

The composition of the invention can be formulated or administered incombination with one or more anticancer drugs to improve clinicalefficacy by reducing cardiotoxicity and cardiac damage side effects ofprolonged use of anticancer drugs. Examples of anticancer agentsinclude, but are not limited to: chemotherapeutic agents (e.g., arsenictrioxide, cisplatin, carboplatin, chlorambucil, melphalan, nedaplatin,oxaliplatin, triplatin tetranitrate, satraplatin, imatinib, nilotinib,dasatinib, and radicicol, an alkylating agent, an anthracycline, anepothilone, a histone deacetylase inhibitor, an inhibitor oftopoisomerase I, an inhibitor of topoisomerase II, a cytoskeletaldisruptor, a kinase inhibitor, a monoclonal antibody, a peptideantibiotic, a nucleotide analog/precursor analog, a retinoid, and avinca alkaloid), immunomodulatory agents (e.g., methotrexate,leflunomide, cyclophosphamide, cyclosporine A, minocycline,azathioprine, antibiotics (e.g., tacrolimus), methylprednisolone,corticosteroids, steroids, mycophenolate mofetil, rapamycin, mizoribine,deoxyspergualin, brequinar, T cell receptor modulators, and cytokinereceptor modulators), antiangiogenic agents (e.g., bevacizumab, suramin,and etrathiomolybdate), mitotic inhibitors (e.g., paclitaxel,vinorelbine, docetaxel, abazitaxel, ixabepilone, larotaxel, ortataxel,tesetaxel, vinblastine, vincristine, vinflunine, and vindesine),nucleoside analogs (e.g., gemcitabine, azacitidine, capecitabine,carmofur, cladribine, clofarabine, cytarabine, decitabine, floxuridine,fludarabine, fluorouracil, mercaptopurine, pentostatin, tegafur, andthioguanine), DNA intercalating agents (e.g., doxorubicin, actinomycin,bleomycin, mitomycin, and plicamycin), topoisomerase inhibitors (e.g.,irinotecan, aclarubicin, amrubicin, belotecan, camptothecin,daunorubicin, epirubicin, etoposide, idarubicin, mitoxantrone,pirarubicin, pixantrone, rubitecan, teniposide, topotecan, valrubicin,and zorubicin), folate antimetabolites (e.g., pemetrexed, aminopterin,methotrexate, pralatrexate, and raltitrexed), mitocans (e.g., sodiumdichloroacetate and 3-bromopyruvic acid), and other targeting agents(e.g., agents that target particular enzymes or proteins involved incancer or agents that target particular organs or types of cancers), andcombinations thereof.

Immunosuppressive Agents

The compositions of the invention can be used in combination with animmunosuppressive agent or a drug that inhibits or prevents activity ofthe immune system. These agents are used to prevent rejection oftransplanted organs and tissues, treat autoimmune diseases, and treatsome non-autoimmune inflammatory disease. Examples of immunosuppressiveagents include, but are not limited to, glucocorticoids (e.g.,hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, beclometasone,fludrocortisones, deoxycorticosterone, and aldosterone), cytostatics(e.g., nitrogen mustards, nitrosoureas, platinum compounds,cyclophosphamide, methotrexate, azathioprine, mercaptopurine,pyrimidine, fluorouracil, and protein synthesis inhibitors,dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin),antibodies (e.g., T-cell receptor directed antibodies (e.g,muromonab-CD3), IL-2 receptor directed antibodies (e.g., basiliximab,and daclizumab), drugs acting on immunophilins (e.g., ciclosporin,tacrolimus, and sirolimus), interferons, opiods, and TNF bindingproteins.

Prevention Drugs for Cardiovascular Diseases

Compositions of the invention can be administered in combination withone or more drugs that are used as secondary prevention drugs forcardiovascular diseases. Examples of preventative drugs include, but arenot limited to, β blockers (e.g., nonselective agents, e.g., alprenolol,carteolol, oxprenolol, sotalol, timolol, e.g., β₁-selective agents,e.g., acebutolol, betaxolol, celiprolol, metoprolol, e.g., β₂-selectiveagents, e.g., butaxamine, e.g., β₃-selective agents, e.g., SR 59230A),statins (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin,mevastatin, pravastatin, simvastatin, and rosuvastatin), fibrates (e.g.,bezafibrate, ciprofibrate, clofibrate, gemfibrozil, and fenofibrate),biguanides (e.g., metformin, phenformin, buformin, and proguanil),antihypertension agents, and/or ACE inhibitors (e.g.,sulfhydryl-containing agents, e.g., captopril, zofenopril, e.g.,dicarboxylate-containing agents, e.g., enalapril, ramipril, quinapril,perindopril, imidapril, e.g., phosphate-containing agents, e.g.,fosinopril).

Anti-Neurodegenerative Drugs

The composition of the invention can be administered in combination withone or more anti-neurodegenerative drugs. Examples ofanti-neurodegenerative drugs include, but are not limited to,acetylcholinesterase inhibitors (e.g., donepezil, galantamine, andrivastigmine), anti-glutamate agent (e.g., amantadine, GABA-ergic,valproic acid), reserpine, tetrabenazine, typical/atypical neuroleptics,tricyclic antidepressants, SSRIs, carbamazepine, baclofen, tizanidine,hydergine, choline, piracetam, and lamotrigine.

Antifibrotic Agents

The compositions of the invention can also be administered incombination with one or more antifibrotic agents. Examples ofantifibrotic agents include, but are not limited to pentoxyphiline,tocopherol, vitamin E, pioglitazone, INT 747, peginterferon 2b,infliximab, ribavirin, glycyrrhizin, candesartan, losartan, irbesartan,ambrisentan, FG-3019, warfarin, insulin, colchicines, peginterferon 2a,etanercept, pirfenidone, nintedanib, and IL-10. Typically, an agent canbe identified as an antifibrotic agent if it possesses one or more ofthe following characteristics: 1.) eliminate the cause(s) of injury andtheir mediators; 2.) reduce inflammation and the immune response; 3.)target specific signaling: receptor-ligand interaction, intracellularsignaling (e.g., the renin-angiotensin system, PPARγ, farnesoid, FXR,PXR, or LXR signaling, or NF-κB signaling); 4.) reduce fibrogenesisand/or inhibit matrix synthesis; and 5.) resolve fibrosis by increasingscar matrix degradation, stimulating apoptosis of stellate cells, orcell transplantation.

The following examples are intended to illustrate, rather than limit,the invention.

EXAMPLES Example 1: Experimental Methods

Reagents

Polyclonal Abs against human calcineurin, α-SMA, phosphorylated(p)Smad3, and total Smad2/3 were purchased from Cell SignalingTechnology (2614S; Danvers, Mass.), Sigma-Aldrich (A2547; St. Louis,Mo.), and Cell Signaling Technology (8769S and 3102S), respectively.Goat polyclonal antibodies against mouse endoglin, Type I collagen, andα-SMA were purchased from R&D Systems (BAF1320), Santa Cruz (SC-25974),and Sigma-Aldrich (A2547), respectively. Rabbit polyclonal antibodies tomouse calcineurin (2614S) were purchased from Cell Signaling. Polyclonalantibodies to mouse pSmad-3 (9520), and pERK-1/2 (SC-134900) werepurchased from Cell Signaling and Santa-Cruz. Polyclonal antibodies tomouse total Smad-3 (SC-101154) and total ERK (SC-135900) were purchasedfrom Santa-Cruz. Sugen (SU5416) was purchased from Sigma-Aldrich. AnIgG1 antibody that binds human and mouse endoglin (TRC105) was kindlyprovided by Tracon Pharmaceuticals, San Diego, Calif. An enzyme linkedimmunosorbent assay (ELISA) kit for the detection of active TGFβ1 levelsin mice was purchased from R&D Systems.

Mouse Model of Pharmacologically Induced Right Ventricular PressureOverload

Animals were treated in compliance with the Guide for the Care and Useof Laboratory Animals (National Academy of Science), and protocols wereapproved by the Tufts Medical Center Institutional Animal Care and UseCommittee (Boston, Mass.). Adult, male, 12- to 14-week-old C57BL/6 WTand congenic Eng+/− mice received once-weekly intraperitoneal injectionsof Sugen and were exposed to either normoxic conditions (room air) orchronic normobaric hypoxia (10% O₂), as previously described in Ciuclanet al., Am J Respir Crit Care Med. 184:1171-1182, 2011. After 5 weeks ofexposure to either Sugen+ Normoxia (Su-Norm) or Sugen+ Hypoxia(Su-Hypox), mice underwent hemodynamic analysis with a RV conductancecatheter (Millar Instruments Inc., Houston, Tex.), as described below,and tissue was then obtained for further analysis.

Mouse Model of Surgically Induced Right Ventricular Pressure Overload

Animals were treated in compliance with the Guide for the Care and Useof Laboratory Animals (National Academy of Science), and protocols wereapproved by the Tufts Medical Center Institutional Animal Care and UseCommittee. Adult, male, 12-14 week old C57BL/6 WT and congenic Eng+/−mice underwent pulmonary artery constriction (PAC) as previouslydescribed in Urashima et al., Heart Circ Physiol. 2008:295:H1351-H1368,2008 and Kapur et al., PLoS ONE. 8:e70802, 2013. Specifically, mice wereintubated using a 24G angiocath and mechanically ventilated (HarvardApparatus) at 95 breaths per minute with a tidal volume of 0.3 mL with2.0-2.5% Isoflurane and 100% flow-through oxygen. Depth of anesthesiawas monitored by assessing palpebral reflex, toe pinch, respirations,and general response to touch. Using sterile technique, a leftthoracotomy was performed to isolate and encircle the main pulmonaryartery using a 7-0 nylon suture that is then tied tightly around apre-sterilized, blunt end needle. After de-airing, the thorax is closedwith layered 6-0 Dexon sutures to eliminate the risk of pneumothorax.Post-operative analgesia is immediately provided with subcutaneousbuprenorphine 0.1 mL, which is continued twice daily and as needed foran additional 72 hours. Severe RVPO was induced by PAC with a 25G needlefor 7 days in WT and Eng+/− mice. To investigate the role of endoglin inRVPO, WT mice received 15 mg/kg of either a neutralizing antibody toendoglin (N-Eng Ab; TRC105; Tracon Pharma) or an IgG1 control antibody(IgG Ab; R&D Systems) via single intraperitoneal injection 1 day priorto and 3 days after induction of severe RVPO. To study the effect ofblocking endoglin activity after induction of RVPO, WT mice wererandomized to receive biweekly IP injections for three weeks of 15 mg/kgN-Eng Ab or IgG control Ab beginning three weeks after induction ofmoderate RVPO using a 23G needle for PAC. The antibody dose was based ona previous clinical study demonstrating effective saturation of endoglinreceptors described in Rosen et al., Clin Cancer Res. 18:4820-4829,2012. After 7 days of severe PAC or 3 to 6 weeks of moderate RVPO, miceunderwent hemodynamic analysis with a RV conductance catheter (MillarInc) as previously described in Kapur et al., PLoS ONE. 8:e70802, 2013.Briefly, mice were anesthetized with 2.0% isoflurane administered via anon-invasive nose-cone. Body temperature was monitored by a rectalthermistor probe and maintained at 37.5° C. with heating pads and acycling heat lamp. In the supine position, the right external jugularvein was surgically isolated. A conductance catheter was advanced intothe right ventricle for pressure-volume loop acquisition as described inKapur et al., PLoS ONE. 8:e70802, 2013. After completion of thehemodynamic study, with the animal still under isoflurane anesthesia,the chest was rapidly opened, and the mouse was euthanized by arrestingthe heart in diastole with 0.3 mL of 1N KCL injected directly into theleft ventricle. The heart was then removed and processed for eitherbiochemical or histologic analyses.

Nuclear Factor of Activated T-Cell Activity In Vivo

Nuclear factor of activated T-cell (NFAT)-luciferase (NFAT-Luc) micewith nine copies of an NFAT-binding site from the interleukin (IL)-4promoter (5′-TGGAAAATT-3′) inserted upstream of the luciferase reportergene, driven by the α-myosin heavy-chain promoter were purchased fromThe Jackson Laboratory (Bar Harbor, Me.). Eng+/−-NFAT luciferasereporter mice were generated by crossing Eng+/− mice with theNFAT-luciferase mice. Severe RVPO was induced by PAC in 10- to12-week-old Eng+/+-NFAT-Luc and Eng+/−-NFAT-Luc. After 7 days of severePAC, RV tissue was then obtained for quantification of luciferaseactivity using firefly luciferase assays that were carried out asfollows: 20 μL of whole RV tissue lysate was added to 100 μL of fireflyluciferase assay buffer (Promega, Madison, Wis.). Samples were placed ina luminometer (Luminoskan Ascent; Labsystems Oy, Helsinki, Finland), andluminescence was determined in triplicate per sample over a 10-secondinterval.

Hemodynamic Assessment of RV Function

All animals underwent terminal hemodynamic evaluation. Right heartcatheterization was performed at the time of sacrifice in all animals.Mice were anesthetized with 2.0% isoflurane administered by anoninvasive nose cone. Body temperature was monitored by a rectalthermistor probe and maintained at 37.5° C. with heating pads and acycling heat lamp. In the supine position, the right common carotid andright external jugular vein were surgically isolated. Silk ties wereplaced at the distal ends of both vessels while overhand loops wereplaced at the proximal ends with 7-0 nylon. A Millar PVR-1035 (MillarInstruments) mouse conductance catheter was used for RV recordings.Before insertion, conductance catheter calibration was performed usingthe cuvette method with freshly heparinized warm blood, then zeroed inwarm saline as previously described in Rockman et al., Proc Natl AcadSci USA. 91:2694-2698, 1994 and Kass et al., Circulation. 73:596-595,1986. A transverse venotomy was performed using iris scissors at theproximal end of the external jugular vein. The PVR-1035 catheter wasadvanced through the superior vena cava and right atrium into the RV,leaving the chest wall intact. Once hemodynamic stability was achieved,steady-state baseline conditions were recorded from the RV. Strokevolume was calculated as end-diastolic minus end-systolic volume.Arterial elastance was calculated under steady-state conditions asend-systolic pressure/stroke volume. Tau, a measure of instantaneousisovolumic relaxation, was calculated using the Glantz method asP(t)=P₀e^(−t/τE)+Pα, where P is pressure at time t, P₀ is the amplitudeconstant, τ_(E) is the Glantz relaxation constant, and Pα is the nonzeroasymptote resulting from pleural and pericardial pressure. RV compliancewas calculated as stroke volume divided by peak RV pressure.Pressure-volume loop acquisition and analysis was performed using IOXsoftware (emka TECHNOLOGIES, Paris, France). After completion of thehemodynamic study, with the animal still under isoflurane anesthesia,the chest was rapidly opened, and the mouse was euthanized by arrestingthe heart in diastole with 0.3 mL of 1 N of KCL injected directly intothe LV. The heart was then removed and processed for either biochemicalor histologic analyses.

Histologic Quantification of Cardiac Hypertrophy and Fibrosis

RV collagen abundance was quantified by picrosirius red staining asdescribed in Georgescu et al., Am J Physiol Cell Physiol.301:C1046-1056, 2011. Cardiomyocyte cross-sectional area was quantifiedas described in Patten et al. J Card Fail. 14:245-253, 2008.

Loss of Function Studies in Cardiac Fibroblasts

Briefly, adult WT and Eng+/− mice were intubated using a 24G angiocathand mechanically ventilated (Harvard Apparatus) at 95 breaths per minutewith a tidal volume of 0.3 mL with 2.0-2.5% Isoflurane and 100%flow-through oxygen. Depth of anesthesia was monitored by assessingpalpebral reflex, toe pinch, respirations, and general response totouch. With the animal still under isoflurane anesthesia, the chest wasrapidly opened, and the mouse was euthanized by arresting the heart indiastole with 0.3 mL of 1N KCL injected directly into the leftventricle. The heart was then removed and processed for isolation ofcardiac fibroblasts, primary culture, and TGFβ1 stimulation aspreviously described in Kapur et al., Circulation. 115:67-75, 2007 andNeuss et al., Cell Tissue Res. 286:145-153, 1996. For neutralizingantibody studies in vitro, mouse cardiac fibroblasts were pretreatedwith 10, 50, or 100 ug/mL of either a N-Eng Ab or control IgG Ab for 24hours in fibroblast basal medium without supplementation prior tostimulation with TGFβ1 (10 ng/mL). After 24 hours, cells were harvestedfor analysis. The antibody dose was based on previous studiesdemonstrating effective neutralization of endoglin activity inendothelium described in Nolan-Stevaux et al., PLoS ONE. 7:e5-920, 2012.

Human RV (RVFB) and LV (LVFB) fibroblasts were isolated from myocardialtissue harvested during cardiac surgery at Tufts Medical Center, andmouse RVFB and LVFB were isolated from WT and Eng+/mice. Fibroblastswere stimulated with TGFβ1 for analysis, as previously described inKapur et al., Circulation 125:2728-2738, 2012; Kapur et al.,Circulation. 115:67-75, 2007; and Neuss et al., Cell Tissue Res.286:145-153, 1996. For calcineurin inhibition studies, human RVFB werepretreated with 5 nM of cyclosporine A (CsA) or vehicle control for 24hours in fibroblast basal medium (FBM) without supplementation, followedby stimulation with TGFβ1 (10 ng/mL) for 24 hours. For TRPC-6 silencingexperiments, 50 μmol/L of siRNA stock was diluted to 5 nmol/L in Optimem(Invitrogen, Carlsbad, Calif.) and combined with 2 μL of Lipofectamine(Invitrogen) diluted in 98 IL of Optimem. After 20 minutes ofincubation, cells were exposed to human TRPC-6 siRNA (Catalog No.:439420; Ambion, Austin, Tex.) or scrambled siRNA (negative control;Catalog No.: 4390844; Ambion). After 48 hours after transfection, cellswere treated with TGFβ1 (10 ng/mL) for 16 to 24 hours, then harvestedfor analysis. For neutralizing Ab studies in vitro, human RVFB and LVFBwere pretreated with 10, 50, or 100 μg/mL of either an N-Eng Ab orcontrol IgG Ab for 24 hours in FBM before stimulation with TGFβ1 (10ng/mL). After 24 hours, cells were harvested for analysis. The Ab dosewas based on previous studies demonstrating effective neutralization ofendoglin activity in endothelium. All RVFB and LVFB stimulation studieswere conducted in triplicate with cells cultured to within three lineagepasses only.

Real-Time Quantitative Polymerase Chain Reaction (RT-PCR)

For all cell-based RT-PCR experiments, total RNA was extracted directlyusing Trizol (Invitrogen), converted to cDNA using a High Capacity cDNAReverse Transcription Kit (Applied Biosystems). For all RT-PCRexperiments, samples were quantified in triplicate using 40 cyclesperformed at 94° C. for 30 sec., 60° C. for 45 sec, 72° C. for 45 secusing an ABI Prism® 7900 Sequence Detection System with appropriateprimers (Table 1) as described in Patten et al. J Card Fail. 14:245-253,2008 and Kapur et al., Circulation 115:67-75, 2007.

TABLE 1 Primer Sequences Mouse Primers Type I collagen ForwardAAG GGT CCC TCT GGA GAA CC (SEQ ID NO: 3) ReverseTCT AGA GCC AGG GAG ACC CA (SEQ ID NO: 4) Calcineurin (CN-PP) ForwardCCACAGGGATGTTGCCTAGTG (SEQ ID NO: 5) Reverse GTCCCGTGGTTCTCAGTGGTA(SEQ ID NO: 6) Endoglin Forward CTG CCA ATG CTG TGC GTG AA(SEQ ID NO: 7) Reverse GCT GGA GTC GTA GGC CAA GT (SEQ ID NO: 8) α-SMAForward GCATCCACGAAACCACCTA (SEQ ID NO: 9) Reverse CACGAGTAACAAATCAAAGC(SEQ ID NO: 10) MYH7 Forward ATG TGC CGG ACC TTG GAA (SEQ ID NO: 11)Reverse CCT CGG GTT AGC TGA GAG ATC A (SEQ ID NO: 12) TRPC-6 ForwardGGC GGC TCT CTA AAG GCT G (SEQ ID NO: 13) ReverseTGG GGT AGT AGC CAT ACG GTG (SEQ ID NO: 14) Human PrimersType I collagen Forward GTC GAG GGC CAA GAC GAA G (SEQ ID NO: 15)Reverse CAG ATC ACG TCA TCG CAC AAC (SEQ ID NO: 16) Calcineurin (CN-PP)Forward TGCATCAATTCTTCGACAGG (SEQ ID NO: 17) ReverseAAGGCCCACAAATACAGCAC (SEQ ID NO: 18) α-SMA Forward CCGACCGAATGCAGAAGGA(SEQ ID NO: 19) Reverse ACAGAGTATTTGCGCTCCGAA (SEQ ID NO: 20) TRPC-6Forward GCCAATGAGCATCTGGAAAT (SEQ ID NO: 21) ReverseTGGAGTCACATCATGGGAGA (SEQ ID NO: 22)Immunoblot Analysis (Western)

Total protein was extracted and quantified from tissue homogenates orcultured cells as described in Patten et al. J Card Fail. 14:245-253,2008 and Kapur et al., Circulation. 115:67-75, 2007. Immunoblot analysiswas then performed as previously described in Patten et al. J Card Fail.14:245-253, 2008 and Kapur et al., Circulation 115:67-75, 2007, usingantibodies for mouse targeted proteins.

Statistical Analysis

Results are presented as mean±standard deviation. Intergroup comparisonswere made with two-factor ANOVA. Repeated measures ANOVA were used asneeded to account for time. All multiple comparisons versus a controlgroup were performed using Dunnett's method. Kaplan-Meier analysis withlog-rank testing was employed for survival analysis. All statisticalanalyses were performed using SigmaStat Version 3.1 (Systat Software,Inc). An alpha level of P<0.05 was considered to indicate a significanteffect or between-groups difference.

Example 2: Reduced Endoglin Expression Preserves RV Function andImproves Survival in RVPO

To explore the functional role of endoglin in RV remodeling Eng+/− micewas studied. Compared to WT, baseline RV endoglin expression was lowerin Eng+/− mice (FIGS. 1A-1B). Severe RVPO was then induced by PAC for 7days in WT and Eng+/− mice. In WT mice, compared to sham controls, PACincreased endoglin levels in the RV, suggesting a direct effect of RVPOon endoglin expression. RVPO also increased endoglin expression inEng+/− mice, but levels were significantly lower compared to WT mice(FIGS. 1A-1B). The functional impact of reduced endoglin levels in RVPOwas then examined. Eng+/− mice demonstrated substantially improvedsurvival (100% versus 58%, respectively, p=0.01) compared with WT miceafter PAC (FIG. 1C). Despite equally increased RV systolic pressure inboth WT and Eng+/− mice after PAC, RV stroke volume decreased in WT, butnot Eng+/− mice (FIG. 1D-1E; Table 2). WT mice also manifest reducedtotal body weight after RVPO, while Eng+/− mice did not (FIG. 1F). Thesefindings suggest that despite identical degrees of RVPO, reducedendoglin expression in Eng+/− mice preserved RV function and improvedsurvival.

TABLE 2 Characterization of Right Ventricular Pressure Overload inWild-Type and Eng+/− Mice induced by PAC, Sugen, or Hypoxia Wild TypeEng^(+/−) Sham (n = 6) PAC (n = 7) Sham (n = 6) PAC (n = 8) Total bodyweight, g 35 ± 2 24 ± 2*  34 ± 4 28 ± 2  RV weight/tibial length, g/mm 1.4 ± 0.1  3 ± 0.1*  1.7 ± 0.3 2.3 ± 0.1 LV weight/tibial length, g/mm  6 ± 0.4  4 ± 0.3*   5 ± 0.2     4 ± 0.3*^(,†) Hemodynamic variables RVsystolic pressure, mm Hg 21 ± 6 50 ± 4*  24 ± 3 46 ± 9  RV end-diastolicpressure, mm Hg  4 ± 2 8 ± 4   2 ± 1 4 ± 2 RV +dp/dt, mm Hg/sec 2358 ±392 3328 ± 1163* 2064 ± 343  3517 ± 1118* RV −dp/dt, mm Hg/sec 2514 ±187 2613 ± 849  2079 ± 341 2715 ± 622* RV stroke volume, μL  9 ± 3 4 ±1*  8 ± 2  7 ± 1^(†) Cardiac output, mL/min  5 ± 1 2 ± 1*  4 ± 1  4 ±1^(†) Heart rate, beats per min 540 ± 62 532 ± 51  509 ± 13 521 ± 24 Wild Type Eng^(+/−) Su-Norm Su-Hypox Su-Norm Su-Hypox Total body weight,g 27 ± 2 27 ± 1  29 ± 2 28 ± 2  RV weight/tibial length, g/mm  1.2 ± 0.41.4 ± 0.4  1.4 ± 0.1 1.5 ± 0.1 LV weight/tibial length, g/mm 4.8 ± 3 4.4 ± 3  4.9 ± 2  5.3 ± 1  Hemodynamic variables RV systolic pressure,mm Hg 23 ± 2 36 ± 2  24 ± 4 34 ± 3  RV end-diastolic pressure (mm Hg)  2± 1 3 ± 1  3 ± 3 2 ± 2 RV +dp/dt, mm Hg/sec 2259 ± 217 3203 ± 456* 2476± 257 2924 ± 156* RV −dp/dt, mm Hg/sec 2162 ± 149 3212 ± 642  2333 ± 4182100 ± 493  RV stroke volume, μL  7 ± 3 8 ± 3  8 ± 1 8 ± 2 Cardiacoutput, mL/min  3794 ± 1827 3898 ± 1670  4150 ± 1345 3995 ± 1529 Heartrate, beats per min 507 ± 37 504 ± 28  514 ± 52 506 ± 53  LV indicatesleft ventricular; RV, right ventricular. *P < 0.01 versus Su-Norm (n =6/group).

Example 3: Reduced Endoglin Expression Limits RV Fibrosis andTGFβ1/Calcineurin Activity in RV Pressure Overload

To study the mechanism underlying improved survival in Eng+/− mice,changes in RV structure were examined. RVPO increased RV mass in WT, notEng+/− mice (Table 2). RV cardiomyocyte cross-sectional area wasincreased in both WT and Eng+/− mice after RVPO, but the degree ofhypertrophy was lower in Eng+/− mice (FIGS. 1G-1H). RVPO also increasedRV fibrosis in WT, but not Eng+/− mice (FIGS. 2A-2B). Consistent withthis observation, collagen levels were increased in WT, but not Eng+/−mice after RVPO (FIG. 2C). These findings suggest that endoglinregulates changes in RV structure in RVPO.

Next, TGFβ1 signaling in RVPO was studied. Despite equally increasedactive TGFβ1 protein levels in WT and Eng+/− mice (FIG. 2D), levels ofpSmad-3 and pERK-1/2 were increased in WT mice, but not Eng+/− mice(FIGS. 1I-1J). Reduced levels of calcineurin mRNA and protein expressionin Eng+/− mice was observed compared to WT after RVPO (FIG. 2E-F).Levels of downstream targets of calcineurin activity including MYH7 andTRPC-6 were also reduced in Eng+/− mice compared to WT after RVPO (FIGS.2G-2H). Levels of α-SMA mRNA were also increased in WT, but not Eng+/−mice after RVPO, indicating reduced fibroblast to myofibroblastconversion in Eng+/− mice (FIG. 2I). These observations suggest thatcanonical and non-canonical TGFβ1 pathways that promote cardiac fibrosisare activated by RVPO and require endoglin. Furthermore, reducedendoglin levels limited RV expression of both calcineurin and α-SMA, keycomponents of myofibroblast transformation, supporting an important rolefor endoglin in RV remodeling.

To further explore whether endoglin regulates calcineurin activity, RVPOwas induced in Eng+/+-NFAT-Luc and Eng+/−-NFAT-Luc mice. RVPO increasedluciferase activity in total RV lysates from Eng+/+-NFAT-Luc, notEng+/−-NFAT-Luc, mice (FIG. 9). These observations suggest that, inaddition to regulating canonical and noncanonical TGFβ1 pathways thatpromote cardiac fibrosis, reduced endoglin levels in the RV limitcalcineurin expression and activity, including myofibroblasttransformation. These findings support an important role forendoglin-mediated regulation of TGFβ1 and calcineurin activity in RVremodeling.

Example 4: Neutralizing Endoglin Activity Prevents RV Fibrosis andImproves Survival in RVPO

To confirm whether calcineurin expression requires endoglin in RVPO, WTmice were pretreated with a N-Eng Ab (TRC105) or control IgG Ab beforePAC. Despite equally increased RV systolic pressures in both groups(Table 3), N-Eng Ab treatment improved survival after 7 days of severeRVPO compared to IgG treatment (FIG. 3A). RV cardiomyocyte crosssectional area was increased in both groups, but a trend towards lowercardiomyocyte hypertrophy was observed in N-Eng Ab treated mice comparedto IgG controls (p=0.09) (FIG. 2J-2K). RV mass was also increased inboth groups, but the degree of hypertrophy was attenuated in N-Eng Abtreated mice after RVPO (Table 1). RV fibrosis was significantly reducedin mice receiving the N-Eng Ab (FIG. 3B-3C) along with reduced Type Icollagen and calcineurin levels (FIG. 3D-3E) compared to the IgG groupafter RVPO. Levels of pSmad-3 and pERK-1/2 were also reduced in theN-Eng Ab group, compared to the IgG group after RVPO (FIG. 2L-2M).Levels of downstream targets of calcineurin activity including MYH7,TRPC-6, and α-SMA mRNA were also reduced in the N-Eng Ab group afterRVPO (FIGS. 3F-3H).

TABLE 3 Characterization of Right Ventricular Pressure Overload inducedby severe PAC in Wild-Type Mice Pre-treated with a Neutralizing AntibodyAgainst Endoglin (N-Eng Ab) or IgG-Isotype Control Antibody (IgG) WildType Wild Type + N-Eng Ab Sham PAC Sham PAC Total body weight, g 29 ± 223 ± 2* 28 ± 1 24 ± 2* RV weight/tibial length, g/mm  1.5 ± 0.01  2.5 ±0.01*  1.5 ± 0.01    1.9 ± 0.01*^(,†) LV weight/tibial length, g/mm    6± 0.01    4 ± 0.01*    6 ± 0.01    5 ± 0.02* Hemodynamic variables RVsystolic pressure, mm Hg 22 ± 3 48 ± 4* 24 ± 3 53 ± 9* RV end-diastolicpressure, mm Hg  4 ± 1 7 ± 4  3 ± 2 4 ± 2 RV +dp/dt, mm Hg/sec 2374 ±429 3189 ± 982  2171 ± 283 4130 ± 563* RV −dp/dt, mm Hg/sec 2419 ± 3042810 ± 891  1963 ± 257 3287 ± 350* RV stroke volume, μL  8 ± 3  4 ± 1* 8 ± 2  5 ± 1* Cardiac output, mL/min 4.3 ± 1  1.8 ± 1*  4.0 ± 1  2.4 ±0.2 Heart rate, beats per min 538 ± 25 548 ± 33  512 ± 59 514 ± 52  PACindicates pulmonary artery constriction; LV, left ventricular; RV, rightventricular. *P < 0.01 versus sham; ^(†)P < 0.01 versus wild-type PAC (n= 6/group).

Example 5: Endoglin is Required for Calcineurin Expression andMyofibroblast Conversion in RV Fibroblasts

The role of endoglin as a regulator of calcineurin expression incultured fibroblasts from the RV (RVFB) and LV (LVFB) of WT and Eng+/−mice was studied. Human RVFB were stimulated with TGFβ1 in the presenceor absence of the calcineurin inhibitor, CsA. Pretreatment withcyclosporine attenuated TGFβ1-mediated increases in protein and mRNAlevels of calcineurin and α-SMA (FIG. 7A through 7C). TGFβ1 stimulationalso increased TRPC-6 mRNA expression in human RVFB, which was preventedby cyclosporine treatment (FIG. 7D). To examine the role of TRPC-6 in RVmyofibroblast transformation, a siRNA against TRPC-6 was used(siTRPC-6), which achieved a greater than 75% knockdown of TRPC-6protein expression in RVFB (FIG. 7E). Silencing TRPC-6 attenuatedTGFβ1-mediated up-regulation of calcineurin and α-SMA in human RVFB(FIG. 7F). These data indicate that TGFβ1 increases expression of TRPC-6and α-SMA in a calcineurin-dependent manner in human RV fibroblasts.

In WT RVFB, TGFβ1 stimulated both calcineurin and α-SMA mRNA expression,which was prevented in Eng+/−RVFB. In contrast, TGFβ1 inducedcalcineurin and α-SMA expression were increased in both WT andEng+/−LVFB (FIGS. 4A-4C). To further explore the dependence ofcalcineurin expression on endoglin in RVFB and LVFB, cells were treatedwith TGFβ1 in the presence of increasing concentrations of the N-Eng Ab.TGFβ1 induced calcineurin and α-SMA protein expression were inhibited bythe N-Eng Ab in RVFB not LVFB (FIG. 4D-4F). In contrast, TGFβ1 inducedprotein levels of pSmad-3 and pERK-1/2 were inhibited by N-Eng Abtreatment in both RVFB and LVFB (FIG. 3I-3J). These findings suggestthat endoglin is required for TGFβ1-induced calcineurin expression andmyofibroblast transformation of cardiac fibroblasts originating from theRV, not LV.

Previous studies of TGFβ1 activity in cardiac remodeling have focused onLV failure; yet, TGFβ1 signaling in the RV remains largely unexplored.The majority of understanding of the mechanisms governing RV remodelingstem primarily from data generated in models of LV failure. However,substantial differences between the RV and LV exist that support thepotential for the two ventricles to have distinct responses to injury,including: (1) the developmental origin of the RV from a heart fielddistinct from the LV; (2) a thin RV free wall with susceptibility toincreased wall stress; (3) a greater dependence of the RV stroke volumeon afterload; and (4) enhanced RV contractile resilience to pressureoverload. In this study, reduced endoglin expression had no effect on LVexpression of calcineurin. Despite all that is known in the LV,regulation of profibrotic signaling in the RV remains poorly understoodand the role of endoglin in the RV has never been studied. These studiesexploring the role of endoglin in the RV response to pressure overloadreveal that, although some similarities exist with the LV, there arealso pathways unique to endoglin's role in the RV. Indeed, endoglinlimited TGFβ1 signaling by Smad3 and ERK1/2 in both ventricles; however,in contrast to previous observations in the LV, endoglin is shown toregulate TGFβ1-induced calcineurin expression and activity in the RV. Itwas uniformly observed that reduced endoglin activity attenuatedcalcineurin expression and activity, as evidenced by reduced levels ofdownstream targets of calcineurin activity, including MYH7 and TRPC-6.

Example 6: Neutralizing Endoglin Activity Reverses RV Fibrosis inEstablished RVPO

To confirm the clinical utility of blocking endoglin activity as anapproach to reduce cardiac fibrosis after established RVPO, WT micesubjected moderate RVPO for 3 weeks were randomized to receive either aN-Eng Ab or control IgG Ab for an additional 3 weeks. After 3 weeks ofmoderate RVPO, total body weight was reduced, while RV mass and systolicpressure were increased and RV stroke volume decreased compared to shamcontrols (Table 4). RV fibrosis, Type I collagen, and calcineurinexpression were also increased compared to sham controls (FIG. 5). Afteran additional 3 weeks (6 weeks total) of moderate RVPO, both IgG andN-Eng Ab treated groups had persistently increased RV mass and RVsystolic pressure with reduced cardiac output. No mortality was observedafter moderate RVPO in either group at any time point (Table 4). RVfibrosis progressively worsened in mice treated with the control IgG Ab,but was significantly reduced in the N-Eng Ab treated group (FIG. 5).Type I collagen and calcineurin protein expression also increasedprogressively in the IgG group, but were reduced in the N-Eng Ab group.These findings confirm that blocking endoglin activity reverses RVfibrosis in chronic RVPO.

TABLE 4 Characterization of Chronic Right Ventricular Pressure OverloadInduced by Moderate PAC in Wild-type mice treated with either aNeutralizing Antibody against Endoglin (N-Eng Ab) or IgG-Isotype ControlAntibody (IgG Ab) PAC 6 weeks + 6 weeks + Sham 3 weeks IgG Ab N-Eng AbTotal body weight, g 31 ± 1  27 ± 2* 27 ± 1* 27 ± 2*  RV weight/tibiallength, g/mm 1.4 ± 0.5  2.7 ± 0.5*  2.7 ± 0.4* 2.5 ± 0.4* LVweight/tibial length, g/mm 5.4 ± 0.5  3.5 ± 0.1*  4.1 ± 0.1* 4.5 ± 0.2*Hemodynamic variables RV systolic pressure, mm Hg 26 ± 1  70 ± 5*  69 ±10* 69 ± 14* RV end-diastolic pressure, mm Hg 1 ± 1 4 ± 2 2 ± 1 2 ± 1 RV +dp/dt, mm Hg/sec 2212 ± 52  4836 ± 929* 4215 ± 674* 4072 ± 875*  RV−dp/dt, mm Hg/sec 2115 ± 64  4171 ± 278* 4345 ± 818* 3916 ± 875*  RVstroke volume, μL 9 ± 2  3 ± 1* 3 ± 2 3 ± 2* Cardiac output, mL/min 4.3± 1  1.5 ± 1*   1.3 ± 0.5* 1.4 ± 0.6* Heart rate, beats per min 500 ±81  592 ± 83  527 ± 69  550 ± 52  PAC indicates pulmonary arteryconstriction; LV, left ventricular; RV, right ventricular. *P < 0.01versus sham (n = 6/group).

Example 7: Reduced Endoglin Expression Preserves RV Function and LimitsRV Fibrosis in a Model of Angio-Obliterative Pulmonary Hypertension

To further explore a functional role for endoglin in RV remodeling, thewell-established model of angio-obliterative pulmonary hypertensioninduced by exposure to hypoxia and the anti-vascular endothelial growthfactor compound, Sugen, was studied in WT, compared to Eng+/− mice. Allmice survived treatment with Sugen+Hypoxia for 5 weeks, and nosignificant change in total body weight, RV or LV weights, RV strokevolume, or cardiac output was observed between groups (Table 2).Increased RV systolic pressure (RVSP) was observed in both WT and Eng+/−mice after 5 weeks of exposure to Sugen+ Hypoxia (FIG. 8A). Nodifference in RV dP/dtmax was observed between WT and Eng+/− groupstreated with Sugen+ Hypoxia, demonstrating a similar response to RVPO inboth types of mice. WT mice exposed to Sugen+ hypoxia developed evidenceof abnormal diastolic RV function, including increased Tau (a measure ofinstantaneous isovolumic relaxation) and decreased RV compliance (FIGS.8B and 8C), whereas Eng+/− mice demonstrated no change in Tau andrelatively preserved RV compliance. To explore the mechanism for thedifferences in RV diastolic function, RV fibrosis and calcineurinsignaling were examined. Exposure to Sugen+Hypoxia increased type Icollagen mRNA expression and histologic levels of collagen abundance inWT, not Eng+/−, mice (FIG. 8D through 8F). Calcineurin, TRPC-6, andα-SMA mRNA levels were increased by Sugen+ Hypoxia in WT, not Eng+/−,mice (FIG. 8G through 8I). These findings suggest that, despiteidentical degrees of RVPO, reduced endoglin expression in Eng+/− micepreserved indices of RV diastolic function, limited RV collagenaccumulation, attenuated up-regulation of calcineurin and TRPC-6, andlimited myofibroblast transformation in the RV.

Example 8: Endoglin Selectively Modulates TRP Channel Expression inResponse to LV or RV Pressure Overload

To explore a functional role for endoglin as a regulator of TRPCexpression in response to RV or LV pressure overload, Eng+/− and Eng+/+mice were exposed to TAC or PAC constriction for 10 weeks. Biventriculartissue was then analyzed by RT-PCR.

After TAC, LV levels of TPRC1 and 6 were increased in both Eng^(+/+) andEng+/− mice compared to sham controls. LV levels of TRPC4 were increasedin Eng+/+, not Eng+/− mice after TAC (FIG. 10A). After PAC, RV levels ofTRPC 1, 3, 4, and 6 were increased in Eng+/+ compared to sham controls.In contrast, chronic RV pressure overload did not increase RV levels ofTRPC 1,3,4, and 6 in Eng+/− mice compared to sham controls (FIG. 10B).After TAC, LV levels of TRPM3 and 7 were increased in Eng+/+ compared tosham controls (FIG. 11A). After PAC, RV levels of TRM3 and 7 wereincreased in Eng+/+ compared to sham controls (FIG. 11B). In contrast,chronic RV pressure overload did not increase RV levels of TRPM3 and 7in Eng+/− mice compared to sham controls (FIG. 11B). After TAC, LVlevels of TRPV2 and 4 were increased in Eng+/+, not Eng+/− mice afterTAC (FIG. 12A). After PAC, RV levels of TRPV2 and 4 were increased inEng+/+ compared to sham controls. In contrast, chronic RV pressureoverload did not increase RV levels of TRPV2 and 4 in Eng+/− micecompared to sham controls (FIG. 12B).

The TRPC family of Ca2+ permeable channels includes 7 members and canincrease intracellular calcium levels ([Ca2+]i), which activatescalcineurin expression in fibroblasts and promotes myofibroblasttransformation. Several previous reports have established that TRPC-6amplifies pathological signaling by participating in a self-propagatingfeed-forward circuit mediated by calcineurin activity and is therefore apotentially important target of therapy in cardiac remodeling (Kuwaharaet al., J Clin Invest. 116:3114-3126, 2006; Davis et al., Dev Cell.23:705-715, 2012; and Berry et al., Circ Res. 109:407-417, 2011). Untilnow, no studies have examined the functional role of endoglin and TRPsignaling pathways in RVPO. Taken together, the data show that pressureoverload induces distinct profiles of TRP expression in the RV and LV ofmice and in some cases, expression of particular TRP channelsspecifically in the RV require full endoglin activity.

Example 9: Endoglin is Required for Regulation of Fibrotic Signaling inthe Lung and Kidney

To determine whether endoglin is an important component in fibroticsignaling, not limited to the RV, fibrotic signaling in lung tissue wasexamined in the context of Eng+/+ and Eng+/− mice. FIG. 13 shows thatendoglin is required for collagen expression in mouse lung tissue. ThePCR result was obtained in mice subjected to two weeks of pulmonaryvenous congestion due to thoracic aortic constriction (TAC) and leftheart failure. The results show that reduced endoglin expressionattenuates increased collagen expression in lung tissue.

In addition, fibrotic signaling in renal tissue was examined in thecontext of Eng+/+ and Eng+/− mice. FIG. 14A shows that endoglin isrequired for collagen expression in mouse renal tissue. The PCR resultwas obtained in mice induced with LV failure by TAC. The results showthat reduced endoglin expression decreases collagen expression in renaltissue. Plasminogen activator inhibitor-1 (PAI-1) expression was alsoanalyzed in renal tissue. PAI-1 is an inhibitor of serin proteases tPAand uPA/urokinase and thus is involved in the regulation offibrinolysis. Excess levels of PAI-1 has been implicated in metabolicsyndrome and various other disease states (e.g., atherothrombosis,obesity, and various forms of cancer). FIG. 14B shows that endoglin isrequired for PAI-1 expression in mouse renal tissue and that reducedendoglin expression decreases PAI-1 expression in renal tissue. Thus,together, the data indicate that endoglin is required for regulation offibrotic signaling and modulation of endoglin activity would be usefulin reducing fibrosis in the context of treating lung disease and kidneydisease.

The central findings in these studies is that endoglin modulates TGFβ1signaling through canonical, noncanonical, and calcineurin-mediatedpathways in the RV, modulates fibrotic signaling in organs, such as thelung or kidney, presumably also through TGFβ1 signaling, and could be atherapeutic target to limit organ fibrosis and improve survival indiseases characterized by RVPO and/or fibrosis. Several findingsreported herein include: (1) Endoglin is necessary for TGFβ1-inducedincrease in expression of TRPC-6 and α-SMA by a calcineurin-dependentmechanism in human RV fibroblasts; (2) TRPC-6 mediates a feedback looppromoting calcineurin expression and myofibroblast transformation inhuman RV fibroblasts that is also dependent on endoglin; (3) in Eng+/−mice exposed to Sugen+ Hypoxia, reduced endoglin activity improved RVdiastolic function, limited fibrosis, and attenuated expression ofcalcineurin, TRPC-6, and α-SMA; (4) in the most severe model of surgicalpressure overload, reduced endoglin activity, induced either by geneticmeans or by treatment with a neutralizing Ab, improved survival, reducedRV fibrosis, and limited TGFβ1 signaling through canonical,noncanonical, and calcineurin-mediated pathways in the RV; (5) in micewith established RV fibrosis, neutralizing endoglin activity reversed RVfibrosis and attenuated expression of both type I collagen andcalcineurin and (6) reduced endoglin expression in the lung and kidneyof mice induced with heart failure attenuates increased collagenexpression and decreases key regulators of fibrinolysis. Given theimportance of calcineurin and TRPC-6 in adaptive and maladaptive cardiacremodeling, these findings identify endoglin as a regulator ofTGFβ1-signaling cascades involved in RV remodeling and further show thattargeting endoglin activity improves RV function in heart failure, lungdisease, and/or kidney disease. Because endoglin plays a critical rolein TGFβ1 signaling, targeting endoglin activity also provides a methodfor controlling pathological wound healing and preventing fibrosisrelated morbidity and mortality in organs generally. Accordingly,endoglin can serve as a therapeutic target to limit organ fibrosis andimprove survival in disease states characterized by RVPO and/orfibrosis.

Other Embodiments

All patents, patent applications, and publications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent patent, patent application, or publication wasspecifically and individually indicated to be incorporated by reference.

The invention claimed is:
 1. A method of treating a fibrotic disease ina human subject in need thereof, the method comprising administering tothe subject a therapeutically effective amount of an interferingribonucleic acid (RNA) that is specific for Endoglin mRNA and thatreduces Endoglin expression, wherein the fibrotic disease is selectedfrom the group consisting of lung fibrosis, kidney fibrosis, and liverfibrosis.
 2. The method of claim 1, wherein the interfering RNA isadministered in combination with an antifibrotic agent.
 3. The method ofclaim 2, wherein the antifibrotic agent is selected from the groupconsisting of: pentoxyphiline, tocopherol, vitamin E, pioglitazone, INT747, peginterferon 2b, infliximab, ribavirin, glycyrrhizin, candesartan,losartan, irbesartan, ambrisentan, FG-3019, warfarin, insulin,colchicines, peginterferon 2a, etanercept, pirfenidone, nintedanib, andIL-10.
 4. The method of claim 1, wherein the fibrotic disease is lungfibrosis and the subject has interstitial lung disease.
 5. The method ofclaim 1, wherein the fibrotic disease is kidney fibrosis and the subjecthas diabetic nephropathy.
 6. The method of claim 1, wherein the fibroticdisease is liver fibrosis and the subject has nonalcoholicsteatohepatitis (NASH).
 7. The method of claim 1, wherein the lungfibrosis is idiopathic pulmonary fibrosis.
 8. The method of claim 1,wherein the interfering RNA is an siRNA.
 9. The method of claim 8,wherein the siRNA comprises a strand having a length of from 21nucleotides to 23 nucleotides.
 10. The method of claim 8, wherein thesiRNA is completely complementary to at least 18 consecutive nucleotidesof an Endoglin mRNA.